Human diabetes-mediating proteins

ABSTRACT

Determining the expression level of certain diabetes-mediating proteins provides an approach both for diagnosing diabetes and for ascertaining a predisposition for developing diabetes.

RELATED APPLICATIONS

This application is the U.S. National Phase of PCT/DK03/00190 filed Mar. 20, 2003 which claims priority from Danish Patent Application No. PA 2002 00431 filed on Mar. 20, 2002, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to human diabetes-mediating proteins, methods of identifying diabetes-mediating proteins, methods for screening for drugs which affect the expression of diabetes-mediating proteins, and therapeutic compounds for the treatment and prevention of diabetes.

BACKGROUND OF THE INVENTION

The development of insulin-dependent diabetes mellitus (IDDM) in man, and in animal models of human disease, is characterised by mononuclear cell infiltration and β cell destruction in the pancreatic islets (insulitis). The mechanisms behind β-cell destruction is not known. Accumulating evidence indicates that the cytokines like interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α) or interferon-γ (IFN-γ) or combinations of them, primarily produced by macrophages and monocytes, may be a mediator of islet β-cell destruction [Mandrup-Poulsen T, Nerup J. New concepts in the pathogenesis of insulin-dependent diabetes mellitus. Contrib Nephrol. 1989; 73:1-14; discussion 14-5].

Animal models of human diabetes include diabetes-prone BB (BB-DP) rats and non-obese diabetic (NOD) mice. 2-Dimensional (2D) gel maps of rat islet proteins have been constructed and used to determine qualitative and quantitative changes in protein synthesis resulting with in vitro exposure of rat islet cells to IL-1β (Andersen et al. (1995) Diabetes 44:400-407; John N E et al., Diabetes. (2000); 49:1819-29. Christensen et al., Autoimmunity. (2000); 32:1-15 and Mose Larsen et al., Diabetes. (2001); 50:1056-63). PCT/IB97/01627 describes diabetes-mediating proteins identified by 2-dimensional gel analysis from rats.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery and identification of human diabetes-mediating (DM) proteins. DM proteins are proteins which are involved in the development of diabetes or in the prevention of diabetes development in a subject at risk for the development of diabetes, and are identified by differential expression during the presence and absence of disease development. The development of diabetes includes all stages which precede the clinically detectable stage.

Accordingly, in one aspect the invention features substantially purified human protein, wherein the protein is present in a human islet cell and exhibits an altered expression during the development of diabetes relative to expression in the absence of diabetes development. The purified diabetes-mediating proteins of the invention are selected from the proteins listed in the Tables (including all modifications (such as biochemically or chemically modified proteins), variants or degradation products thereof), and especially in the Tables 6, 7, and 8. In a related aspect, the invention features an isolated protein selected from the group consisting of IEF spots 8, 370, 473, 524, 535, 551, 651, 656, 909, 1013, 1186, 1353, 1400, 1477, 1549, 1629, 1685, 1689, 1707, 1715, 1766, 1800, 1902, 1935, 2041, 2079, 2354, 2382, 2408, 2411, 13652, and 14098 (as listed in Table 6), NEPHGE spots 26, 35, 60, 76, 85, 128, 130, 171, 187, 188, 195, 243, 270, 421, 449, 508, 509, 532, 558, 560, 609, 719, 729, 829, 836, 837, 3879, and 6600 (as listed in Table 7) and IEF spots 122SPI, 123SPI, 126SPI, 130SPI, 135SPI, 140SPI, 160SPI, 218SPI, 248SPI, 277SPI, 304SPI, 314SPI, and 338SPI (as listed in Table 8). The expression of one or more of these proteins (including all modifications, variants or degradation products thereof) may be used to estimate the risk of a test subject for the development of diabetes by obtaining a biological sample from the test subject, and determining the level of expression of one or more of the above listed proteins, wherein an altered expression of one or more of the above-listed proteins may indicate risk of disease development.

In another aspect, the invention features a database of human islet cell proteins, which proteins are identified as human islet cell proteins exhibiting altered protein expression when exposed to a combination of one or more cytokines, relative to a non-exposed human islet cells. In one embodiment, the cells are exposed to IL-1β; in another embodiment, the cells are exposed to a mixture of cytokines comprising one of (i) IFNγ and TNFα, (ii) IFNγ and TNFα, and (iii) IL-1β, IFNγ, and TNFα. In a specific embodiment, a database of human diabetes-mediating proteins is generated by exposure of cells to a combination of IL-1β, IFNγ, and TNFα. In a more specific embodiment, the invention features a database of proteins identified as human islet cell proteins exhibiting altered expression when human islet cells are exposed to 150 pg/ml IL-1β, 1000 U/ml IFNγ, and 5000 U/ml TNFα. This is referred to as ‘cytokine mix’ throughout this document and in the tables.

Proteins exhibiting an altered expression relative to control cells comprise the database of the proteins listed in Tables 1, 2 and 3 and shown in FIGS. 1, 2 and 3. In more specific embodiments, the invention provides subsets of proteins from the protein database comprising the proteins listed in Tables 1, 2 and 3. The database subsets include marker proteins that are diagnostically useful for predicting and/or assessing the risk of a subject for development of diabetes. Accordingly, in one aspect, the invention provides a subset of marker proteins selected from the proteins listed in Table 1 and/or Table 2 and/or Table 3 including all their variants, post-translational modifications, degradation products and peptides derived there from and homologous proteins. In a more specific embodiment, the database comprises 5 or more, 10 or more, 15 or more, or 25 or more of the proteins listed in Table 1, Table 2 and Table 3.

In a related aspect, the invention features a database of secreted human islet cell proteins, comprising secreted human islet cell proteins exhibiting altered expression when exposed to IL-1β, IFNγ, and TNFα, relative to a non-exposed human islet cells. In a specific embodiment, the protein database is comprised of the proteins listed in Table 3 and shown in FIG. 3. These proteins include the proteins identified as 12SPI, 63SPI, 79SPI, 81SPI, 82SPI, 83SPI, 122SPI, 123SPI, 126SPI, 130SPI, 135SPI, 140SPI, 160SPI, 168SPI, 213SPI, 215SPI, 215SPI, 218SPI, 248SPI, 258SPI, 258SPI, 259SPI, 277SPI, 304SPI, 314SPI, 320SPI, 338SPI, and 1157SPI. In more specific embodiments, the database comprises a sub-population of the secreted proteins listed in Table 3. In even more specific embodiments, the database comprises 15 or more, 10 or more, and 5 or more of said proteins.

In another related aspect, the combinations of proteins may be between the cellular proteins (listed in Tables 1 and 2) and the secreted proteins (listed in Table 3).

As shown in Tables 1, 2 and 3 and FIGS. 1, 2 and 3, a number of the diabetes-mediating proteins are identified by correspondence to previously identified proteins by mass spectrometry. Proteins not corresponding to previously identified proteins are characterized herein by their mass spectroscopy spectrum. Novel non-secreted human diabetes-mediating proteins characterized by molecular weight, pI, and the mass spectroscopic characteristics are further grouped in Table 6 for proteins identified on IEF gels, and in Table 7 for proteins identified on NEPHGE gels. For some proteins it was not possible to obtain a mass spectrum and so they are only characterised by their isoelectric point and molecular weight and position in the gel electrophoretograms (FIGS. 1, 2 and 3).

As shown in Table 3 for secreted diabetes-mediating proteins, a number of the secreted diabetes-mediating proteins are identified by correspondence to previously identified proteins by correspondence of pI and molecular weight. Novel secreted proteins, that is proteins not corresponding to previously identified proteins, are characterized herein by mass spectroscopy spectrum (Table 8).

The diabetes-mediating proteins of the invention are useful in drug screening assays for identifying compounds capable of modulating the development of diabetes, useful as therapeutic agents for the treatment or prevention of diabetes, and useful as targets of therapeutic agents capable of preventing or ameliorating diabetes by modulating the expression of the diabetes-mediating protein.

Changes in the expression of specific DM proteins are diagnostically useful as indicative of the development of diabetes and for predicting the course of its development (prognosis). Accordingly, in one aspect the invention features a method for diagnosing the development of diabetes by measuring an increase or a decrease in protein expression in one or more proteins selected from the group consisting of the non-secreted and/or secreted diabetes-mediating proteins listed in Tables 1-3. Changes in protein expression are measured in a test subject suspected of developing diabetes or at risk for the development of diabetes and are expressed relative to protein expression in a normal non-diabetes control. In a preferred embodiment, changes of combinations of one or more of the proteins of Tables 1-3 is indicative of the development of diabetes. In a more preferred embodiment, changes of a combination of at least 5 of the proteins of Tables 1-3 is indicative of the development of diabetes. In an even more preferred embodiment, changes of a combination of at least 10 of the proteins of Tables 1-3 is indicative of the development of diabetes.

The invention provides identified diabetes-mediating proteins which may be further characterized as protective or deleterious proteins, as described in PCT/IB97/01627, the text of which application is specifically incorporated herein by reference. A protective protein is one which prevents, inhibits, or slows the development of diabetes in a subject at risk for diabetes, and a deleterious protein is one that causes the development of diabetes, increases the risk of development of diabetes, or decreases the time required for the development of diabetes in a subject at risk for developing diabetes. A deleterious protein is also a protein that prevents or interferes with the expression of a protective protein.

The invention includes a substantially purified protective or deleterious diabetes-mediating protein, and polynucleotide sequence which encodes the diabetes-mediating protein of the invention.

In one aspect, the invention features an assay for screening compounds which effect the expression of one or more diabetes-mediating proteins. In specific embodiments, the invention provides an assay for identifying a compound capable of inducing or enhancing the expression of an endogenous protective protein, and thus to delay or inhibit the development of diabetes. In another specific embodiment, the assay method of the invention is useful for identifying a compound capable of suppressing or inhibiting the expression of a deleterious diabetes-mediating protein, thus delaying or inhibiting the development of diabetes.

In a related aspect, the invention provides an assay for identifying a compound which modulates the activity of a diabetes-mediating protein, e.g., an agonist, an antagonist, or by blocking a post-translational step required for activation of a diabetes-mediating protein. Changes in the expression of specific DM proteins are useful in a screening method for identifying compounds capable of modulate the expression of DM proteins. A compound which modulates the expression of one or more diabetes mediating proteins is useful as a potential therapeutic in the treatment or prevention of diabetes. Accordingly, in one aspect the invention features an assay method for identifying compounds capable of modulating the expression of diabetes-mediating proteins having the steps of contacting a test compound with a cell or tissue expressing one or more diabetes-mediating proteins, and determining the effect of the test compound on the expression of one or more diabetes-mediating proteins. Determination of the effect of a compound may be conducted by a variety of methods known to the art, including hybridization to probes or other oligonucleotides, antibody recognition, e.g., immunodiffusion, immunofluorescence, ELISA, RIA, blotting, immunoprecipitation, immunoelectrophoresis, or chromatography, and electrophoresis. A compound capable of increasing the expression of one or more proteins selected from the group consisting of the diabetes-mediating proteins listed in Tables 1, 2 and 3 and decreasing the expression of one or more proteins selected from the list consisting of the diabetes-mediating proteins listed in Tables 1, 2 and 3 is a candidate therapeutic agent for the prevention or treatment of diabetes. Changes in protein expression are determined relative to expression in the absence of the test compound.

In another aspect, the invention provides a therapeutic method for preventing diabetes in a subject at risk for diabetes or of ameliorating the symptoms of diabetes in a diabetic subject by administering a therapeutically effective amount of a protective diabetes-mediating protein. Preferably the subject is a human. Also included in the invention is gene therapy by providing a polynucleotide encoding a protective diabetes-mediating protein. The invention further includes a therapeutic method for preventing and/or treating diabetes by administering an effective amount of a polynucleotide which inhibits the in vivo expression of a deleterious diabetes-mediating protein. Candidate therapeutic compounds are selected from the proteins of Tables 1, 2, and 3, homologues and derivatives thereof and mimics thereof.

In a related aspect, the invention provides a therapeutic method of preventing and/or treating diabetes in a subject at risk for diabetes by administering a therapeutically effective amount of a compound capable of suppressing or reducing the expression of an endogenous deleterious diabetes-mediating protein. In another embodiment, the invention provides a therapeutic method of preventing and/or treating diabetes by administering a therapeutically effective amount of a compound capable of inducing or enhancing the expression of an endogenous protective diabetes-mediating protein. In a related aspect, the invention provides a therapeutic method of preventing and/or treating diabetes in a subject at risk for diabetes by administering a therapeutically effective amount of a compound capable of modulating the activity of a diabetes-mediating protein, e.g., as an agonist, an antagonist, or by preventing the activation of a diabetes-mediating protein. The therapeutic method of the invention includes ex vivo methods known to the art for providing the therapeutic agent to a subject in need thereof.

An object of the invention is to identify human proteins which mediate diabetes onset.

Another object of the invention is to provide human diabetes-mediating proteins which are useful in assays for identifying test compounds capable of preventing, delaying, or ameliorating diabetes in a subject.

Another object of the invention is to provide human diabetes-mediating proteins which are useful in assays for identifying test compounds capable of causing, accelerating or worsening diabetes in a subject, indicating that the test compound would not be suitable as a pharmaceutical compound.

These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the diabetes-mediating gene(s) and protein(s), assay method and from the claims.

DETAILED DISCLOSURE OF THE INVENTION

A first aspect of the invention relates to a method for diagnosing diabetes in a human, the method comprising determining the presence or level of expression of at least one marker protein in a biological sample from the human, wherein the marker protein is selected from the group consisting of any one of the proteins disclosed in Tables 1, 2 and 3, and FIGS. 1, 2, and 3 and marker proteins which are modifications and derivatives of marker proteins of Table 1, 2 or 3, so as to have at least 80% homology with marker proteins of Table 1, 2 or 3, wherein pI is the isoelectric point of the marker protein as determined by isoelectric focusing, an up-arrow means that the protein is up-regulated in exposed cells, a down-arrow means that the protein is down-regulated in exposed cells and the molecular weight (Mol. Wt in kDa) is determined on a polyacrylamide gel.

Furthermore, the applications of the method comprises preferably at least 2, at least 5, or even more preferably at least 10 of said proteins because the modification in expression of 2 or more, 5 or more or 10 or more are increasingly statistically-reliable indicators for the application of these markers.

A further aspect relates to a method for diagnosing diabetes in a human, the method comprising determining the presence or level of expression of at least one marker protein in a biological sample from the human, an further comprising establishing the increased expression of at least one marker protein (an up-regulated marker protein) or establishing the decreased expression of at least one marker protein (a down-regulated marker protein) selected from the group consisting of proteins or combinations of up- and down-regulated marker proteins.

The invention further relates to a method of treating diabetes by the up-regulation of a down-regulated protein, the down-regulation of an up-regulated protein, or combinations thereof. That is to say that the invention relates to a method of treating diabetes in a human comprising altering the expressing of a marker protein of Table 1, 2 or 3 shown also in FIGS. 1, 2 and 3. Furthermore, the invention relates to method of treating diabetes in a human comprising administering a marker protein of Table 1, 2 or 3, a nucleotide sequence coding for a marker protein of Table 1, 2 or 3, an antibody for a protein of Table 1, 2 or 3, a nucleic acid fragment capable of binding to a marker protein of Table 1, 2 or 3, or a compound capable of binding to a marker protein of Table 1, 2 or 3 to said human.

In the course of identifying marker proteins as marker proteins for diabetes, said marker proteins either previously not known to be associated with diabetes or their level of expression not known in diabetes (down- or up-regulated), the present investigators have now isolated proteins previously not identified, and certainly not associated with diabetes. In connection to these novel proteins, the invention further relates to a protein selected from the group consisting of proteins of Table 1, 2 and 3 and proteins with at least 80% homology therewith.

A further aspect relates to the use of novel proteins and proteins of Table 1, 2 and 3 as markers or indicators for diabetes as well as to the use of known proteins whose presence, absence or prevalence has previously not been associated with diabetes. The changes in protein expression and patterns of protein expression are considered to be important markers for diagnosis, prognosis and therapeutic applications and targets.

The method of the present invention may be further used to determine the predisposition in a human for diabetes, the method comprising determining the presence or relative level in a biological sample from the human of at least one marker protein wherein the marker protein being indicative of a predisposition for having diabetes is selected from the group consisting of the proteins disclosed in the tables 1, 2 and 3 and marker proteins further consisting of modifications and derivatives of marker proteins of Table 1, 2 or 3, so as to have at least 80% homology with marker proteins of Table 1, 2 or 3.

A method for diagnosing the predisposition in a human for diabetes, may comprise determining the increased expression in a biological sample from the human of at least one marker protein selected from the a biological sample from the human, said marker protein selected from the group consisting of proteins of Table 1, 2 and 3, establishing the decreased expression of at least one marker protein (a down-regulated marker protein) in a biological sample from the human, establishing the increased expression of at least one marker protein, or establishing combinations of increased and decreased expression of the marker proteins. Thus, the determination of whether a protein is up-regulated or down-regulated serves as useful indicators of diabetes susceptibility. The pattern of up and down regulation may also serve as an indicator. That is to say that the level of expression of more than one protein is established and the pattern of expression of a grouping of proteins is used as an indicator.

In a suitably embodiment, at least one marker protein is selected from the group consisting of one or more proteins present in a significantly lower or significantly higher amount on a polyacrylamide gel of proteins from said biological sample in relation to a control, one or more proteins present on a polyacrylamide gel of proteins from said biological sample and absent on polyacrylamide gel of proteins of a control, one or more proteins absent on a polyacrylamide gel of proteins from said biological sample and present on polyacrylamide gel of proteins of a control.

Similarly, with regards to a method of treating diabetes, a single protein may be targeted for therapy or a grouping of proteins may be targeted. The level of expression of these targeted proteins may be altered or the proteins themselves may be interfered with in order to alter their activity. Thus, an interesting embodiment of a method of treating diabetes in a human comprises altering the expressing of at least one marker protein of Table 1, 2 or 3.

The invention thus further relates to a method of treating diabetes in a human comprising administering at least one marker protein of Table 1, 2 or 3, a derivative, homologue or mimic thereof, a nucleotide sequence coding for a marker protein of Table 1, 2 or 3, an antibody for a protein of Table 1, 2 or 3, a nucleic acid fragment capable of binding to a marker protein (or its corresponding gene) of Table 1, 2 or 3, or a compound capable of binding to a marker protein (or its corresponding gene) of Table 1, 2 or 3 to said human.

A method of preventing or delaying the onset or of diabetes in a human according to the present invention may comprise administering a marker protein of Table 1, 2 or 3, a derivative, homologue or mimic thereof, a nucleotide sequence (such as DNA, cDNA, RNA, PNA homologues or mimics thereof) coding for a marker protein of Table 1, 2 or 3, an antibody for a protein of Table 1, 2 or 3, a nucleic acid fragment (such as DNA, cDNA, RNA, PNA, homologues or mimics thereof) capable of binding to a marker protein of Table 1, 2 or 3, or a compound capable of binding to a marker protein of Table 1, 2 or 3 (or its corresponding gene) to said human.

Thus a particularly interesting aspect of the present invention relates to a pharmaceutical composition which comprises a substance which is capable of regulating the expression of a nucleic acid fragment coding for at least a part of a protein of Table 1, 2 or 3, or at least one marker protein in Table 1, 2 or 3, an antibody for a protein of Table 1, 2 or 3, a nucleic acid fragment capable of binding to a marker protein of Table 1, 2 or 3, or a compound capable of binding to a marker protein of Table 1, 2 or 3 to said human.

The invention further relates to a method of determining the likelihood of an agent having a therapeutic effect in the treatment of diabetes comprising determining the level of expression of one or more proteins of Table 1, 2 or 3 before and after exposing a test model to said agent and comparing said levels.

In the testing of compounds, knowledge about the activity or target of an agent is useful for understanding the therapeutic activity of said agent and may assist in improving the desired therapy. The developments of the present investigators allows for a method of determining the effect of a compound in the treatment of diabetes comprising determining the level of expression of proteins of one or more proteins of Table 1, 2 or 3 and to a method of determining the level of effect or level of activity of a compound used in the treatment of diabetes comprising determining the level of expression of one or more proteins of Table 1, 2 or 3 before and after exposing a test model to said agent.

Thus, the invention further relates to a method for determining the physiological effect of a substance, the method comprising using a mammal which has been established to be an individual having a high likelihood of having diabetes or a genetic predisposition for having diabetes by use of a method according to the invention, the method comprising administering the substance to the individual and determining the effect of the substance. The present investigators anticipate that a method of determining the nature or cause of diabetes in a human having or susceptible to said disease comprising establishing the level of expression of a protein of Table 1, 2 or 3 in relation to a model serves for understanding the disease and potential therapies.

Each of the methods of the present invention relate to the use of a protein according to Table 1, 2 or 3 or having least 80% homology therewith and/or are post-translationally modified. Similarly, the newly identified proteins of Table 1, 2 or 3 further encompass proteins with at least 80% homology therewith, which are or are not post-translational modification products of these proteins.

The invention further relates to a nucleic acid fragment comprising a nucleotide sequence which codes for a peptide defined in Table 1, 2 or 3 as well as to a nucleic acid fragment which hybridizes with said nucleic acid fragment or a part thereof. The use of said nucleic acid fragment may serve to detecting the presence of a peptide of Table 1, 2 or 3.

The invention further relates to an antibody able to bind to a protein (or any part of its post-translational modification) defined in Table 1, 2 or 3. The antibody may be a polyclonal antibody or a monoclonal antibody. The use of an antibody may serve for detecting the presence of a peptide shown in Table 1, 2 or 3.

An interesting aspect of the present invention relates to a test kit for diagnosing diabetes or a genetic predisposition for diabetes in a mammal, comprising:

-   a) a binding mean which specifically binds to at least one marker     protein shown in Table 1, 2 or 3 (or any part of its     post-translational modification) or an antibody for a protein of     Table 1, 2 or 3, a nucleic acid fragment capable of binding to a     marker protein of Table 1, 2 or 3 (or any part of its     post-translational modification), or a compound capable of binding     to a marker protein of Table 1, 2 or 3 (or any part of its     post-translational modification) to said human; -   b) means for detecting binding, if any, or the level of binding, of     the binding means to at least one of the marker proteins or at least     one of the peptides or at least one of the nucleic acid fragments,     and -   c) means for correlating whether binding, if any, or the level of     binding, to said binding means is indicative of the individual     mammal having a significantly higher likelihood of having diabetes     or a genetic predisposition for having diabetes.

It should be noted that the detection of any combination of more than one of the markers would be expected to make the analysis an even more reliable indicator for the disease related to diabetes. Thus, a method for diagnosing or determining the predisposition of at least one disease related to diabetes comprising determining the presence, activity, concentration and/or level of expression of a combination of two markers would be preferred and three or more markers (e.g. at least 4, 5, 6 or 7 markers) would be strongly preferred. It is analogously suggested that treatment with more than one compound (e.g. at least 2, 3, 4, 5, 6 or 7 compounds) according to the invention (e.g. more than one compound chosen from the group consisting of: a polypeptide, a nucleic acid fragment or an antibody according to the invention), said compounds combined being able to affect the level of more than one marker protein, would make the treatment of the disease even more efficient.

Before the present human diabetes-mediating proteins and genes and assay methodology used in the assay are described, it is to be understood that this invention is not limited to particular assay methods, diabetes-mediating proteins and genes, test compounds described, as such methods, genes and preparations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “diabetes-mediating protein” or “a diabetes-mediating protein” include mixtures of such diabetes-mediating proteins, reference to “the formulation” or “the method” includes one or more formulations, methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

The term “diabetes” includes insulin-dependent diabetes mellitus (IDDM, juvenile diabetes or T1D) and type II diabetes (adult-onset diabetes, T2D). The term “diabetes-related diseases” includes such conditions as obesity, circulatory deficiencies, insulin-resistance, syndrome X, diabetic retinopathy, diabetic neuropathy, and the involvement of advanced glycation end products (AGE) in neuropathy and atherosclerosis.

The term “protein” includes proteins, polypeptides, and peptides which are chains of amino acids, including all post-translational modifications (e.g., processing and truncations, glycosylations or phosphorylations) which often play decisive roles in modulating protein function. The term also encompasses natural proteins as well as synthetic or recombinant proteins, polypeptides, and peptides. The term “diabetes-mediating protein” means a protein which is involved in the development of diabetes. A diabetes-mediating protein is a protein which exhibits an altered expression during the development of diabetes, that is, a protein which is up- or down-regulated, or whose expression is modulated up or down, during the development of diabetes, as compared to the expression of the same protein in the absence of the development of diabetes. In the present invention, a diabetes-mediating protein is identified as a human islet cell protein which exhibits altered expression as a result of exposure of a human islet cell to one or more cytokines, relative to the expression in a control cell which is not exposed to one or more cytokines.

The protein may be chemically or biochemically modified by being phosphorylated, methylated, sulphylated, glycosylated or by the addition of any form of lipid or fatty acid, ubiquitin or any other large side groups or by containing additional amino acids or any other forms of modification (of which there are over 200 known). These modifications occur at specific sites on the protein and a particular modification at one site can have different effects as the same modification at a different site on the same protein. They can be reversible in the cell where they are used for example to turn on and off enzymes and so the proteins can exist in a variety of forms-each with an associated activity level for each of the proteins functions. Furthermore the polypeptide may be cleaved e.g. by processing at its N- or C-termini to remove signal peptides or be spliced to remove an internal sequence. Examples of many of these can be found in the protein databases like EXPASY and there exist an ever growing range of tools to predict these modifications and their function. Since it is estimated that each protein in man is modified on average 10 times, it is expected that the majority of the proteins identified here are modified in some way or another. Their apparent isoelectric point and molecular weight has thus been given in tables 1, 2 and 3 so that they can be compared to the theoretical values to indicate what effects the modification has had on the protein.

The term “substantially pure,” when referring to a polypeptide, means a polypeptide that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. A substantially pure diabetes-mediating protein is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, diabetes-mediating protein. A substantially pure diabetes-mediating protein can be obtained, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a diabetes-mediating protein, by recovery after electrophoresis of natural or recombinant cells or expression systems, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The term “protein” also encompasses derivatives, analogues and mimetics of the above mentioned polypeptides. Such a derivative, analogue and mimetic preferably have the same activity, e.g. the same kind of enzymatic activity, as the polypeptide which it is derived from. The derivative, analogue or mimetic can have a lower level activity, the same level or preferably, a higher level of activity than the parent polypeptide.

The term “at least one” (e.g. at least one compound or at least one marker protein) encompasses the integers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 etc. It should be understood that a single marker protein can be used, but it can be advantageous to use more than one marker protein in methods of the invention. That is to say that the level of expression of more than one protein is established and the pattern of expression of a grouping of proteins is used as an indicator. Obviously the reliability of identification of diabetes increases as the number in the group increase.

A “peptide mimetic” is a molecule that mimics the biological activity of a peptide but is no longer peptidic in chemical nature. By strict definition, a peptidomimetic is a molecule that no longer contains any peptide bonds (that is, amide bonds between amino acids). However, the term peptide mimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems, which are similar to the biological activity of the peptide. The present invention encompasses peptidomimetic compositions which are analogs that mimic the activity of biologically active peptides according to the invention, i.e. the peptidomimetics can be used for treatment of diabetes related diseases. The peptidomimetic of this invention are preferably substantially similar in both three-dimensional shape and biological activity to the peptides or active sites of such as set forth above.

Alternatively, the mimetic can be an ‘antimimetic’. In other words, a molecule that can fit into and block the active site of the protein, or bind to binding sites or sites of interaction with other biological molecules and so interfere with the function of the protein. Most current drugs are of this type. Such antimimetics that are capable of interacting with the polypeptides of the invention are encompassed by the present invention.

There are clear advantages for using a mimetic or an antimimetic of a given peptide rather than the peptide itself, because peptides commonly exhibit two undesirable properties: (1) poor bioavailability; and (2) short duration of action. Peptide mimetics or antimemetics offer an obvious route around these two major obstacles, since the molecules concerned are small enough to be both orally active and have a long duration of action. There are also considerable cost savings and improved patient compliance associated with peptide mimetics, since they can be administered orally compared with parenteral or transmucosal administration for peptides. Furthermore, peptide mimetics are much cheaper to produce than peptides. Finally, there are problems associated with stability, storage and immunoreactivity for peptides that are not experienced with peptide mimetics.

Thus peptides described above have utility in the development of such small chemical compounds with similar biological activities and therefore with similar therapeutic utilities. The techniques of developing peptidomimetics are conventional. Thus, peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure. The development of peptidomimetics can be aided by determining the tertiary structure of the original peptide by NMR spectroscopy, crystallography and/or computer-aided molecular modelling. These techniques aid in the development of novel compositions of higher potency and/or greater bioavailability and/or greater stability than the original peptide [Dean (1994), BioEssays, 16: 683-687; Cohen and Shatzmiller (1993), J. Mol. Graph. 11: 166-173; Wiley and Rich (1993), Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15: 124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98, all incorporated herein by reference]. Once a potential peptidomimetic compound is identified, it may be synthesized and assayed using the diagnostic assay described herein or an appropriate disease suppressor assay [see, Finlay et al. (1983), Cell, 57: 1083-1093 and Fujiwara et al. (1993), Cancer Res., 53: 4129-4133, both incorporated herein by reference], to assess its activity.

Thus, through use of the methods described above, the present invention provides compounds exhibiting enhanced therapeutic activity in comparison to the polypeptides described above. The peptidomimetic compounds obtainable by the above methods, having the biological activity of the above named peptides and similar three dimensional structure, are encompassed by this invention. It will be readily apparent to one skilled in the art that a peptidomimetic can be generated from any of the modified peptides described previously or from a peptide bearing more than one of the modifications described previously. It will furthermore be apparent that the peptidomimetics of this invention can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.

The term “expression” is meant to include not only the physical expression of a protein, but also as a measure of the activity of an expressed protein. For example, a protein can be expressed as an inactive form, which is activated by phosphorylation. While the actual expression of the protein has not changed, its effective expression (activity) has been modified. On a gel, the change in activity may be measured as the change in expression of a modified form of the protein. By “altered protein” or “altered protein expression” is meant proteins whose expression is increased (“up regulated”), decreased (“down regulated”), inhibited (i.e., turned off), or induced (i.e., turned on) during the development of diabetes.

The term “diabetes-mediating gene or polynucleotide” means genetic material encoding a protein, peptide, or protein fragment which encodes an intact or fragment of a diabetes-mediating protein. The term includes any gene from any species which encodes a diabetes-mediating protein. A diabetes-mediating gene or polynucleotide may be naturally occurring or partially or wholly synthetic.

By the terms “nucleic acid fragment” “polynucleotide”, “nucleic acid sequence” and the like are understood any nucleic acid molecule including DNA, RNA, LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length, e.g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e.g. in DNA therapy, or for use in a method for producing a polypeptide according to the invention, a molecule encoding at least a part of the polypeptide is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being optionally inserted into a vector. When the nucleic acid molecule is used as a probe, as a primer or in antisense therapy, a molecule having a length of 10-100 is preferably used. According to the invention, other molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides (or nucleotide derivatives). It should be understood that these numbers can be freely combined to produce ranges.

As used herein, an “isolated” polynucleotide is a polynucleotide that is not immediately contiguous (i.e., covalently linked) with either of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally-occurring genome of the organism from which the polynucleotide is derived. The term therefore includes, for example, a recombinant polynucleotide which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences. The isolated and purified polynucleotide sequences of the invention also include polynucleotide sequences that hybridize under stringent conditions to the polynucleotide sequences specified herein.

The term “stringent conditions” means hybridization conditions that guarantee specificity between hybridizing polynucleotide sequences. One skilled in the art can select posthybridization washing conditions, including temperature and salt concentrations, which reduce the number of nonspecific hybridizations such that only highly complementary sequences are identified (Sambrook et al. (1989) in Molecular Cloning, 2d ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., hereby specifically incorporated by reference). For instance, such conditions are hybridization under specified conditions, e.g. involving presoaking in 5×SSC and prehybridizing for 1 h at about 40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 μM ATP for 18 h at about 40° C. (Sambrook et al (1989) op cit.). The isolated and purified polynucleotide sequences of the invention also include sequences complementary to the polynucleotide encoding a diabetes-mediating protein (antisense sequences) and ribozymes. In the present application, the hybridization between polynucleotide sequences is preferably conducted under stringent conditions.

The term “sequence identity” (or “sequence homology”) indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to best possible fit possible with the insertion of gaps or alternatively truncation at the ends of the protein sequences. The sequence identity can be calculated as (N_(ref)−N_(dif))100/N_(ref), wherein N_(dif) is the total number of non-identical residues in the two sequences when aligned and wherein N_(ref) is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (N_(dif)=2 and N_(ref)=8). A gap is counted as non-identity of the specific residue (s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (N_(dif)=2 and N_(ref)=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson W. R and D. J. Lipman (1988) PNAS USA 85:2444-2448). In one aspect of the invention, alignment is performed with the sequence alignment method Clustal W with default parameters as described by Thompson J., et al Nucleic Acids Res 1994 22:4673-4680. Alternatively, the degree of homology between two nucleic acid sequences is determined by using GAP version 8 from the GCG package with standard penalties for DNA: GAP weight 5.00, length weight 0.300, Matrix described in Gribskov and Burgess, Nucl. Acids Res. 14(16); 6745-6763 (1986), and the degree of homology between two amino acid sequences is determined by using GAP version 8 from the GCG package (Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711, USA) with standard penalties for proteins: GAP weight 3.00, length weight 0.100, Matrix described in Gribskov and Burgess, Nucl. Acids Res. 14 (16); 6745-6763 (1986).

A preferred minimum percentage of sequence homology is at least 70%, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.

By the term “database” of proteins is meant a collection of proteins selected by possession of at least one common characteristic. The term “database” may be substituted by other terms designating a collection of proteins, including the term “library” or “array.” The present invention provides a database of human islet cell proteins which exhibit, in one embodiment, the common characteristic of an altered expression when exposed to a specific combination of cytokines.

Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations thereof such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The invention also relates to the use of a polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described in the literature exemplified by Lowry, D. B. et al 1999, Nature 400: 269-71.

A monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide of the invention in an immuno assay, or a specific binding fragment of said antibody, is also a part of the invention. The antibodies can be produced by methods known to a person skilled in the art. The polyclonal antibodies can be raised in a mammal, for example, by one or more injections of a polypeptide according to the present invention and, if desired, an adjuvant. The monoclonal antibodies according to the present invention may, for example, be produced by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be produced by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described by McCafferty et al, Nature, 348:552-554 (1990), for example. Further methods for producing antibodies are described in the literature, e.g. in U.S. Pat. No. 6,136,958.

In diagnostics, treatment or testing, an antibody, a nucleic acid fragment and/or a polypeptide of the invention can be used either alone, or as a constituent in a composition. Such compositions are known in the art, and comprise compositions in which the antibody, the nucleic acid fragment or the polypeptide of the invention is coupled, preferably covalently, to at least one other molecule, e.g. a label (e.g. radioactive or fluorescent) or a carrier molecule.

The present invention is further directed to methods for using the compounds described above to therapeutically and/or prophylactically treat a patient for a diabetes related disease.

The methods of the present invention include the steps of: a) incorporating one or more of the compounds of the present invention in a suitable pharmaceutical carrier; and b) administering either a therapeutically effective amount or a prophylactically effective amount of the compound or compounds incorporated in the carrier to a patient.

The term “suitable pharmaceutical carrier” refers to any carrier known in the pharmaceutical arts for administration of compounds to a patient. Any suitable pharmaceutical carrier can be used according to the present invention, so long as compatibility problems do not arise.

Administration of an effective dosage to a patient can be accomplished by parenteral injection, such as intravenously, intrathecally, intramuscularly or intra-arterially. The compounds can also be administered orally or transdermally, or by any other means known to those skilled in the art, e.g. by means of an inhalator or a nasal spray. Oral administration is presently preferred.

As used herein, the term “therapeutically effective amount” refers to that amount of one or more of the compounds of the present invention required to therapeutically treating a patient. Such treatment is appropriate for subjects having a diagnosed diabetes related disease. Similarly, the term “prophylactically effective amount” refers to that amount of one or more of the compounds of the present invention needed to prophylactically treat a patient. Such treatment is appropriate for subjects who, for example, have not yet established any clinical symptoms of a diabetes related disease. It could be advantageous to start a prophylactic treatment as soon it is determined that the subject is in risk for developing a diabetes related disease, e.g. by means of a determination of a predisposition for diabetes by having an altered level of markers. It is known that certain markers for IDDM (e.g. GAD₆₅ and other autoantibodies) can be detected at least 8 years before the onset of the clinical symptoms.

As will be appreciated by a person skilled in the art, the dosage of compound given, the route of administration and the duration of therapy will be dependent not only the type of compound and its effectiveness in treating the disease but also upon the individual being treated, taking into consideration such factors as the body weight of the patient, other therapies being employed to treat the patient, and the condition, clinical response and tolerance of the patient. Dosage, administration, and duration of therapy can be determined by one skilled in the art upon evaluation of these and other relevant factors.

General Aspects of the Invention

Human Diabetes-Mediating Proteins and Polynucleotides

The invention provides human diabetes-mediating proteins, that is, proteins identified as involved in or effected during the development of diabetes. Diabetes-mediating proteins are characterized as proteins whose expression is altered during the development of diabetes relative to their expression in the absence of the development of diabetes. The present disclosure identifies diabetes-mediating proteins from a 2-dimensional gel database of human pancreatic islet cell proteins. Diabetes-mediating proteins include protective diabetes-mediating proteins and deleterious diabetes-mediating proteins. The diabetes-mediating proteins are identified by exposing human islet cells to cytokines which are known or accepted to be involved in the selective destruction of islet cells which precedes the eventual development of diabetes. The invention provides human diabetes-mediating proteins identified through the use of 2-dimensional gels to compare control and cytokine stimulated islets to identify which proteins respond, identifying the proteins which play a role in the cell response. Interlink analysis can be used to define functional groups of proteins and their regulation (e.g., by kinase phosphorylation or other post-translational modifications).

Protective Diabetes-mediating Proteins

The invention provides substantially purified protective diabetes-mediating proteins (“protective proteins”) characterized as capable of protecting against development of diabetes in a subject at risk for the development of the disease or ameliorating or reducing the symptoms of diabetes in a subject suffering from diabetes. The protective protein of the invention may act directly to protect against diabetes, or may act indirectly by inducing or increasing the synthesis of a second protective protein or by reducing or inhibiting the synthesis of a deleterious protein. The invention further includes amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95% and most preferred at least 98% identity to the fully length amino acid sequence of a human diabetes-mediating protein. Percent homology or identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds. (1979) Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

The invention further includes polynucleotide sequences encoding the diabetes-mediating proteins of the invention, including DNA, cDNA, PNA and RNA sequences. It is also understood that all polynucleotides encoding all or a portion of a diabetes-mediating protein are also included herein, as long as they encode a polypeptide with the diabetes-mediating activity. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, such a polynucleotide may be subjected to site-directed mutagenesis. The polynucleotide sequences of the invention also include antisense sequences. Antisense sequences include sequences synthesized with modified oligonucleotides. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the diabetes-mediating polypeptide is encoded by the nucleotide sequence is functionally unchanged.

Deleterious Diabetes-mediating Proteins

Deleterious diabetes-mediating proteins (“deleterious proteins”) are characterized as enhancing the development of or increasing the risk of a subject developing diabetes.

Method for Identifying a Diabetes-mediating Protein

Two-dimensional gel electrophoresis (2-DGE) is a particularly effective tool for separating mixtures of proteins (e.g. Andersen et al. (1995) Diabetes 44:400-407; John N E et al., Diabetes. (2000); 49:1819-29. Christensen et al., Autoimmunity. (2000); 32:1-15 and Mose Larsen et al., Diabetes. (2001); 50: 1056-63). Cell protein extracts are put onto a gel, and the individual proteins are separated first by charge and then by size. The result is a characteristic picture of as many as 1,000 to 5,000 spots, each usually a single protein. Resolution is improved by increasing gel size, and by enhancing the sensitivity through the use of radiolabel methods, silver staining, and the reduction in thickness of the gels to 1.5 mm and less. A further significant improvement in resolution can be obtained by running first dimension gels covering narrow pH ranges (e.g. 1.5, 1 pH units or less). As described in the Examples below, single proteins recovered from 2D gels can be identified by mass spectrometry to obtain a trypsin cleavage pattern as well as the precise molecular weight of each peptide. These observed values are then used to search in DNA and protein databases to determine if matches exist to previously identified proteins. Identity can be determined from a known protein or deduced from high homology to a known protein. When 2D gel electrophoresis is used to separate and identify protein spots which exhibit an altered synthesis during development of diabetes, an identified protein spot is excised from the gel and digested with trypsin to produce peptides. The peptides are recovered from the gel and subjected to mass spectroscopy (matrix assisted laser desorption/ionization mass spectrometry)(MALDI) and the resulting MS-profiles are analyzed against the computerized MS-profiles of all sequences found in the public sequence databases, as well as against propriety sequence information. If any matches to previously cloned sequences are obtained, information about the corresponding gene and encoded protein is collected. When an identified diabetes-mediating protein does not match a previously cloned protein, the protein may be microsequenced to obtain partial amino acid sequence information by methods known to the art. Proteins (when available in sufficient quantities) may also be partially sequenced by for example nano-electrospray tandem mass spectrometry where particular peptides are fragmented in the gas phase and the molecular weights of the fragments used to derive part of the amino acid sequence. Once partial sequence information is available then it is also possible to search in the cDNA or EST (expressed sequence tag) databases in addition to those mentioned previously.

Based upon results obtained from database searches or amino acid sequencing, specific or degenerate primers are constructed and used to screen rat and human islets libraries or first-strand cDNA by PCR is used to clone partial sequences of the corresponding cDNA. The obtained sequences are then used to obtain full-length coding regions either by 5′-race PCR or by conventional hybridization screening techniques, followed by expression of the recombinant protein (Karlsen et al. (1991) Proc. Natl. Acad. Sci. USA 88:8337-8341; Karlsen et al. (1994) in: Insulin secretion and pancreatic beta-cell research, Flatt, P. R., ed., Smith-Gordon, USA; Chapter 64, pp. 1-9; Karlsen et al. (1995) Diabetes 44:757-758).

Diabetes-mediating proteins can be isolated in a variety of ways known to the art, including purification from biological material, expression from recombinant DNA (see above). Conventional method steps include extraction, precipitation, chromatography, affinity chromatography, and electrophoresis. For example, cells expressing a diabetes-mediating protein can be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation. Diabetes-mediating proteins may alternatively be isolated by immunoprecipitation with the use of specific antibodies.

Use of Diabetes-Mediating Proteins for Screening of Compounds Capable of Effecting the Expression of a Diabetes-Mediating Protein

Assay methods provided by the invention are useful for screen compounds capable of effecting the expression of a diabetes-mediating protein, and thus the development of diabetes in a mammal. One model for screening drugs capable of effecting the expression of one or more diabetes-mediating proteins is the administration of compounds suspected of having beneficial effects (including antisense oligonucleotides) to cells in culture. Useful cells are RIN, transfected, or islet cells. The effects of the test compound on protein expression may then be assayed by 2D gel electrophoresis. Another screening model is an in vivo method with the use of a mammal at risk for development of diabetes. Briefly, a mammal with an increased risk for diabetes (e.g., diabetes-prone BB rat or NOD mouse) is exposed to a test compound, and the effect of exposure to the test compound on the development of diabetes determined.

The development of diabetes may be monitored throughout the developmental period by determining the expression of one or more diabetes-mediating proteins and comparing by comparing the time of disease onset with expression and timing in the absence of disease development. Determining the expression of one or more diabetes-mediating proteins includes the diabetes-mediating protein itself, a post-translational modification product, and/or diabetes-mediating protein degradation product. In one embodiment, activation of a diabetes-mediating protein is determined by measuring the level of the diabetes-mediating protein expression in a test sample. A suitable test sample includes a body fluid, such as blood, urine, or cerebrospinal fluid, or fluid derived from it, such as plasma or serum. In a specific embodiment, the level of protein expression in a test sample is measured by Western blot analysis. The proteins present in a sample are fractionated by gel electrophoresis, transferred to a membrane, and probed with labeled antibodies specific for the protein(s). In another specific embodiment, the level of diabetes-mediating protein expression is measured by Northern blot analysis. Polyadenylated [poly(A)⁺] mRNA is isolated from a test sample. The mRNA is fractionated by electrophoresis and transferred to a membrane. The membrane is probed with labeled cDNA. In another embodiment, protein expression is measured by quantitative PCR applied to expressed mRNA.

In yet another aspect, the invention provides for methods for identifying compounds capable of suppressing or reducing the expression of an endogenous deleterious protein, as well as methods for preventing and/or treating diabetes by administering a therapeutically effective among of a compound capable of suppressing or reducing the expression of an endogenous deleterious protein.

The diabetes-mediating proteins of the invention are also useful to screen reagents that modulate diabetes-mediating protein activity. Accordingly, in one aspect, the invention features methods for identifying a reagent which modulates diabetes-mediating protein activity, by incubating a cell expressing a diabetes mediating protein with the test reagent and measuring the effect of the test reagent on diabetes-mediating protein synthesis, phosphorylation, function, or activity. When activation of a diabetes-mediating protein is via phosphorylation, the test reagent is incubated with the diabetes-mediating protein and with either gamma-[³²P] or [³³P]-labeled-ATP (or other mono-nucleotides), or [³²P] or [³³P]-pyrophosphate (phosphoric acid) or [³⁵S]-methionine, and the rate of phosphorylation determined. In another embodiment, the test reagent is incubated with a cell transfected with an diabetes-mediating protein polynucleotide expression vector, and the effect of the test reagent on diabetes-mediating protein transcription is measured by Northern blot analysis. In a further embodiment, the effect of the test reagent on diabetes-mediating protein synthesis is measured by Western blot analysis using an antibody to the diabetes-mediating protein in still another embodiment, the effect of a reagent on diabetes-mediating protein activity is measured by incubating diabetes-mediating protein with the test reagent, [³²P]-ATP (or other radiochemicals mentioned above), and a substrate in the diabetes-mediating protein pathway. All experiments would be compared against a normal labelling of cells with [³⁵S]-methionine to determine modulation of protein expression. The rate of substrate phosphorylation is determined by methods known in the art.

The term modulation of diabetes-mediating protein activity includes agonists and antagonists. The invention is particularly useful for screening reagents that inhibit deleterious protein activity. Such reagents are useful for the treatment or prevention of diabetes.

Therapeutic Applications

The invention provides methods for preventing and/or treating diabetes in a human by administering a therapeutically effective amount of a protective diabetes-mediating protein. Preferably the mammal is a human subject at risk for diabetes.

Drug Screening Using Identified Diabetes-mediating Proteins and Related Diabetes Therapeutic Agents

In a drug-screening assay of the invention, identified protective or deleterious diabetes-mediating proteins are used to identify test compounds capable of effecting their expression. Test compounds so identified are candidate therapeutic agents for preventing, ameliorating, or delaying the onset of diabetes in a subject at risk.

A test therapeutic compound which effects the expression of a diabetes-mediating proteins can be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof, or any combination thereof, which can be detectably labeled as for labeling antibodies, as described herein. Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds.

A therapeutic compound is identified in the drug screening assay of the invention through its ability to induce or enhance the expression of a protective protein, such that disease onset is prevented or delayed in a subject at risk for the development of diabetes. A candidate therapeutic compound is also identified by its ability to prevent or decrease the expression of a deleterious protein, such that disease onset is prevented or delayed in a subject at risk for the development of diabetes. A therapeutic nucleic acid as a therapeutic compound can have, but is not limited to, at least one of the following therapeutic effects on a target cell: inhibiting transcription of a deleterious protein DNA sequence; inhibiting translation of a deleterious protein RNA sequence; inhibiting reverse transcription of an RNA or DNA sequence corresponding to a deleterious protein; inhibiting a post-translational modification of a protein; inducing transcription of a DNA sequence corresponding to a protective protein; inducing translation of an RNA sequence corresponding to a protective protein; inducing reverse transcription of an RNA or DNA sequence corresponding to a protective protein; translation of the nucleic acid as a protein or enzyme; and incorporating the nucleic acid into a chromosome of a target cell for constitutive or transient expression of the therapeutic nucleic acid. Therapeutic effects of therapeutic nucleic acids can include, but are not limited to: turning off a defective gene or processing the expression thereof, such as antisense RNA or DNA; inhibiting viral replication or synthesis; gene therapy as expressing a heterologous nucleic acid encoding a therapeutic protein or correcting a defective protein; modifying a defective or under-expression of an RNA such as an hnRNA, an mRNA, a tRNA, or an rRNA; encoding a drug or prodrug, or an enzyme that generates a compound as a drug or prodrug in pathological or normal cells expressing the diabetes-mediating protein or peptide; and any other known therapeutic effects. Also included in the invention is gene therapy by providing a polynucleotide encoding a protective diabetes-mediating protein. The invention further includes a method for preventing diabetes by administering an effective amount of a polynucleotide which inhibits the in vivo expression of a deleterious diabetes-mediating protein.

In the therapeutic method of the invention, a therapeutic compound is administered to a human patient chronically or acutely. Optionally, a protective protein is administered chronically in combination with an effective amount of a compound that acts on a different pathway than the therapeutic compound. The therapeutic method of the invention can be combined with other treatments for diabetes or with methods for the management of diabetes. Therapeutic formulations are described in PCT/IB97/01627, which text is specifically incorporated herein by reference for the description of therapeutic formulations and the administration of therapeutic compounds known to the art, including conventional and gene therapeutic techniques.

Identification and Characterization of Human Diabetes-Mediating Proteins

As described in the Example below, human islet of Langerhans isolated from the pancreas of organ donors were cultured under standard culture conditions in RPMI 1640 medium in the presence of recombinant cytokines as follows:

-   -   A=culture medium alone;     -   B=culture medium+150 pg/ml interleukin-1β (IL-1β) (equivalent to         60 U/ml);     -   C=culture medium+1500 pg/ml IL-1β;     -   D=culture medium+1000 U/ml−IFN-γ+5000 U/ml tumour necrosis         factor-α (TNFα);     -   E=150 pg/ml IL-1β+1000 U/ml IFNγ+5000 U/ml TNFα.

After incubation for 20 hr under standard cell culture conditions, cells were labelled in the presence of [³⁵S]- methionine for 4 hours. Proteins were isolated from both cells and culture media. Proteins obtained from culture media represent proteins secreted as a result of exposure to none, one or more cytokines. Protein samples were analyzed by 2-D gel electrophoresis and mass spectroscopy.

For analytical gels, used for the identification of proteins of altered expression level, 6 individual experiments were performed with 150 islets in each experiment. This allowed the construction of a composite Image of each culture condition. Thus, comparison and statistical evaluation between the control condition (A) and any of the other conditions of cytokine exposure (B-E) by this means allowed identification of a set of proteins in the islets as well as secreted to the medium, that were significantly up- or down-regulated compared to the control situation. In order to MS identify the protein behind the spots identified as up-or downregulated in the analytical gel analysis described above, quantitative/preparative gels with 100,000 islets per conditions were made (to get sufficient amount of protein in each spot to allow MS identification). Thus, two sets of 100.000 islets were cultured in the absence of cytokines (A) or in the presence of the 3 cytokines (E), to increase the chance to have all protein spots of interest available for the MS identification.

Experimental

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the proteins, genes and assays of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1

Materials and Methods

Reagents

Ketamin was purchased from Park-Davis (Barcelona, Spain), xylazin from Bayer (Leverkusen, Germany), and Temgesic® from Reckitt and Colemann (Hull, UK). RPMI 1640, Hanks' balanced salt solution (HBSS), and DMEM were purchased from Gibco, Paisley, Scotland. RPMI 1640 contained 11 mmol D-glucose, and was supplemented with 20 mM HEPES buffer, 100,000 IU/l penicillin and 100 mg/l streptomycin. Authentic recombinant human IL-1β was provided by Novo Nordisk Ltd. (Bagsvaerd, Denmark) having a specific activity of 400 U/ng.

Other reagents used: 2-mercaptoethanol, bovine serum albumin (BSA), Tris-HCl, Tris base, glycine, (Sigma, St. Louis, USA); trichloracetic acid (TCA), phosphoric acid, NaOH, glycerol, n-butanol, bromophenol blue, sodium nitroprusside (SNP), H₃PO₄ and NaNO₂ (Merck, Darmstadt, Germany); filters (HAWP 0.25 mm pore size) (Millipore, Boston, USA); RNAse A, DNAse I (Worthington, Freehold, N.J., USA); [³⁵S]-methionine (SJ 204, specific activity: >1.000 Ci/mmol, containing 0.1% 2-mercaptoethanol), Amplify® (Amersham International, Amersham, UK); urea (ultra pure) (Schwarz/Mann, Cambridge, Mass., USA); acrylamide, bisacrylamide, TEMED, ammonium persulphate (BioRad, Richmond, Calif., USA); ampholytes: pH 5-7, pH 3.5-10, pH 7-9, pH 8-9.5 (Pharmacia, Uppsala, Sweden); Nonidet P-40 (BDH, Poole, UK); ampholytes: pH 5-7 and sodium dodecyl sulphate (Serva, Heidelberg, Germany); agarose (Litex, Copenhagen, Denmark); ethanol (absolute 96%) (Danish Distillers, Aalborg, Denmark); methanol (Prolabo, Brione Le Blanc, France); acetic acid (technical quality, 99% glacial) (Ble & Berntsen, Århus, Denmark) and X-ray film (Curix RP-2) (AGFA).

Human Samples

Human islets were isolated (Andersen et al. (1995) Diabetes 44:400-407; John N E et al., Diabetes. (2000); 49:1819-29. Christensen et al., Autoimmunity. (2000); 32:1-15 and Mose Larsen et al., Diabetes. (2001); 50: 1056-63) and provided by the Diabetes Research Inst., Division of Cellular Transplantation, Miami, Fla., USA. After a few days of culture, the islets were exposed to the experimental conditions detailed above. At the end of the experiments the washed islets and culture/labelling media were either directly (for preparative gels) or following lysis in sample buffer (for analytical gels) immediately stored at −80° C. prior to 2D-gel analysis.

Cytokine Challenge

The prepared human islet cells were cultured in five different experimental conditions: Group A was cultured in culture medium alone; Group B in culture medium+150 pg/ml IL-1β (equivalent to 60 U/ml); Group C in culture medium+1500 pg/ml IL-1β; Group D in culture medium +1000 U/ml IFNγ+5000 U/ml TNFα; and Group D in 150 pg/ml IL-1β+1000 U/ml IFNγ+5000 U/ml TNFα. The cells were incubated for 20 hours at 37° C. under standard culture conditions, followed by 4 hr incubation with [³⁵S]-methionine. Cells were collected for 2-D gel electrophoresis and mass spectrophotometric analysis. Labelled media was collected from each experimental group for analysis of secreted proteins.

Sample Preparation

Whereas the 150 islets/condition used for the analytical part were directly lysed in lysis buffer, the larger number of islets (100,000/condition) cultured under conditions A and E to be used for the preparative gels and MS identification as described above, were crushed in a mortar and resuspended in 100 ml DNAse I/RNAse A solution and lysed by freeze/thawing twice. After the second thawing, the samples were left on ice for 30 min for the digestion of nucleic acids and then freeze dried overnight. The samples were dissolved by shaking in 120 ml lysis buffer (8.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol and 2% ampholytes, pH range 7-9) for a minimum of 4 h before 2D-gel separation.

Determination of [³⁵S]-methionine Incorporation

The amount of [³⁵S]-methionine incorporation was quantitated by adding 10 mg BSA (0.2 mg/ml H₂O) as a protein carrier to 5 ml of a 1:10 dilution of each sample in duplicate, followed by 0.5 ml of 10% TCA. This was left to precipitate for 30 min at 4° C. before being filtered through 0.25 mm HAWP filters. The filters were dried and placed into scintillation liquid for counting.

2-D Gel Electrophoresis

The procedure has been described earlier (O'Farrell et al. (1977) Cell 12:1133-1142). Briefly, first dimension gels contained 4% acrylamide, 0.25% bisacrylamide and ampholytes. Equal numbers of counts (10⁶ cpm) of each sample were applied to the gels. In case of lower amounts of radioactivity it was necessary to regulate the exposure time of the gel so that comparable total optical densities were obtained. The samples were analyzed on both isoelectric focusing (IEF; pH 3.5-7) and non-equilibrium pH-gradient electrophoresis (NEPHGE; pH 6.5-10.5) gels. Second dimension gels contained 12.5% acrylamide and 0.063% bisacrylamide and were run over-night. After electrophoresis, the gels were fixed and treated for fluorography with Amplify® before being dried. The gels were placed in contact with X-ray films and exposed at −70° C. for 3 to 40 days. Each gel was exposed for at least 3 time periods to compensate for the lack of dynamic range of X-ray films. Some gels were dried immediately and the gel exposed to a phosphorimaging plate to capture the image. After exposure for from 3 to 15 days (1 exposure per gel because the phosphorimager's dynamic range is in excess of 10⁶), the plates were read in an AGFA ADC 70 and the images exported for analysis as 16 bit files.

Determination of MW and pI

Molecular weights of the proteins were determined by interpolation using values from known proteins. Landmark proteins were identified on gels by one or several of the following techniques: immunoblotting, immunoprecipitation, mass spectrometry, microsequencing or peptide mapping.

Computer Analysis of Fluorographs

Computer analysis was performed using the BioImage® program (version 6.1) on a Sunsparc workstation. First, the fluorographs or autoradiographs were scanned and spots identified and quantitated by the BioImage® program. Next, anchor points were placed on the gel (same spot in each gel was assigned the same anchorpoint), and the computer was asked to match the gels. After computer matching, manual editing was performed to ensure correct spot boundary identification, correct matching of computer found spots and quantitation of spots not found initially by the computer program. Finally, data were extracted for calculations in the Excel® spreadsheet (Microsoft). To avoid the presence of duplicate spots in the IEF and NEPHGE subgroups, overlapping spots in either the basic part of IEF gels or in the acidic part of NEPHGE gels were omitted from analysis.

Statistical Analysis

Student's t test was applied and P<0.01 was chosen as level of significance.

EXAMPLE 2 Characterization of Diabetes-Mediated Proteins

Mass Spectroscopy

In situ digestion is performed on at least one gel plug including at least one protein spot in at least one gel according to the present invention. Gels are prepared by a modification of the method of Rosenfeld et al. (1992) Anal. Biochem. 203:173-179, as described in Fey et al. (1997) Electrophoresis 18:1-12, both of which references are herein specifically incorporated by reference. Briefly, gels are quickly stained and destained. The protein of interest is obtained by cutting a gel band containing the protein with a scalpel and storing in eppendorf tubes with UHQ water at −20° C. The protein is digested by washing the gel plug for at least 1 hour in 40% acetonitrile/60% digestion buffer until the coomassie stain is removed.

This wash removes coomassie stain, gel buffers, SDS and salts. If necessary the wash can be repeated. The gel plug is then dried in a vacuum centrifuge for 20-30 min. until the plug shrinks and becomes white on the surface. Drying time depends on the size and thickness of the gel plug. Trypsin (or the enzyme being used is dissolved in digestion buffer and 5 mls added to the gel plug (depending on the amount of the protein in the gel to be analyzed (0.1 mg)). Additional digestion buffer is added until the gel plug is almost covered by buffer in the bottom of the tube, approximately 10 ml. The gel plug is then incubated at 37° C. for 6 hours or overnight, then incubated with 70-100 ml 60% acetonitrile/40% water for 2-6 hours to extract the peptides. The extraction may be repeated to increase recovery. The extract is then lyophilized and dissolved in 30% acetonitrile/2%TFA before analyzing by MALDI-MS.

EXAMPLE 3 Quantitative Gel Analysis

Analysis of the two-dimensional gels described above resulted in identification of a database of human diabetes-mediating proteins listed in Tables 1-8. These proteins have been identified by treating human islets with either 1500 pg/ml IL-1β or with a mixture of cytokines (150 pg/ml IL-1β (beta interleukin), 1000 U/ml IFNγ (gamma interferon), and 5000 U/ml TNFα (alpha tumour necrosis factor).

Table 1 lists all cellular proteins identified as exhibiting a statistically significant altered expression when [³⁵S]-methionine labelled human islets are treated with cytokines and the proteins analysed on IEF gels. In each of the tables 1, 2, and 3 IL-1β 1500 refers to the stimulation by 1500 pg/ml IL-1β. ‘Cytokine mix’ refers to the stimulation induced by 150 pg/ml IL-1β, 1000 U/ml IFNγ, and 5000 U/ml TNFα. An arrow pointing up indicates that the protein is expressed in higher amounts in the treated cells; and a downward pointing arrow, the reverse.

Table 2 lists all cellular proteins identified as exhibiting a statistically significant altered expression when [³⁵S]-methionine labelled human islets are treated with cytokines and the proteins analysed on NEPHGE gels.

Table 3 lists all secreted proteins identified as exhibiting a statistically significant altered expression or secretion when [³⁵S]-methionine labelled human islets are treated with cytokines and the proteins analysed on IEF gels.

Table 4 lists human islet cell proteins identified as exhibiting an altered expression upon cytokine challenge when analysed on IEF gels (cf. Table 1) and provides the mass spectroscopy molecular weight values obtained for the protein fragments. These proteins have been identified by reference to proteins from other species recorded in publicly available databases (including amino acid and nucleotide sequences).

Table 5 lists human islet cell proteins identified as exhibiting an altered expression upon cytokine challenge when analyzed on NEPHGE gels (cf. Table 2) and provides the mass spectroscopy molecular weight values obtained for the protein fragments. The proteins have been identified by reference to proteins from other species recorded in publicly available databases (including amino acid and nucleotide sequences).

Table 6 lists novel human islet cell proteins identified from IEF gels (cf. Table 1) which did not correspond to any known protein and provides the mass spectroscopy molecular weight values obtained for the protein fragments.

Table 7 lists novel human islet cell proteins identified from NEPHGE gels (cf. Table 2) which did not correspond to any known protein and provides the mass spectroscopy molecular weight values obtained for the protein fragments.

Table 8 lists proteins secreted by human islet cells identified from IEF gels (cf. Table 3) which did not correspond to any known protein and provides the mass spectroscopy molecular weight values obtained for the protein fragments.

FIG. 1 shows a two dimensional gel image of human islets that have been labelled with [³⁵S]-methlonine and the proteins separated according to the procedures described herein. Proteins mentioned in the text or tables are marked in for reference. Approximate molecular weight scale is given on the right. The image is presented with the acidic side to the right and covering the nominal pH range of 4 to 7.

FIG. 2 shows a two dimensional gel image of human islets that have been labelled with [³⁵S]-methlonine and the proteins separated according to the procedures described herein. Proteins mentioned in the text or tables are marked in for reference as is an indicative molecular weight scale. The image is presented with the acidic side to the right and covering the nominal pH range of 6.5 to 10.5.

FIG. 3 shows a two dimensional gel image of human proteins secreted by islets that have been labelled with [³⁵S]-methionine and the proteins separated according to the procedures described herein. Proteins mentioned in the text or tables are marked in for reference as is an indicative molecular weight scale. The image is presented with the acidic side to the right and covering the nominal pH range of 4 to 7.

TABLE 1 HUMAN ISLET CELL PROTEINS CHARACTERISED BY MS. IDENTIFIED, IDENTIFIED BY RELATION TO ANOTHER SPECIES AND UNIDENTIFIED PROTEINS. Gene I|1β Cyt. Spot # Name Identifier 1500 Mix Name Protein Obs. pI Mol. Wt 8 ↓ Novel spectrum see table 6 4.79 249,415 191 HS74 P34932 ↓ Heat shock 70 kD protein 4 4.95 127,654 (HSP70RY) HSPA4. 267 EZRI P15311 ↓ Ezrin (P81) (Cytovillin) (Villin-2) 6.34 69,286 VIL2 297 ↓ Novel unidentified protein 5.90 76,695 370 ↓ Novel spectrum see table 6 5.15 88,075 473 ↓ ↓ Novel spectrum see table 6 6.31 69,863 504 HS7C P11142 ↑ Heat shock cognate 71 kD protein 5.96 62,425 HSPA8 OR HSC70 OR HSP73 524 ↑ Novel spectrum see table 6 5.78 63,041 535 ↑ Novel spectrum see table 6 5.60 70,495 551 ↑ Novel spectrum see table 6 5.53 70,096 554 ↓ Novel unidentified protein 5.40 66,003 651 ↓ Novel spectrum see table 6 4.67 78,015 656 ↓ Novel spectrum see table 6 4.65 69,924 677 ↑ ↑ Novel unidentified protein 6.84 68,176 699 DHAX P49419 ↓ ↓ Inosine-5′-monophosphate dehydrogenase 6.46 61,913 2 (EC 1.1.1.205) (IMP dehydrogenase 2) (IMPDH-II) (IMPD 2) IMPDH2 OR IMPD2. 699 IMD2 P12268 ↓ ↓ Inosine-5′-monophosphate dehydrogenase 6.46 61,913 2 (EC 1.1.1.205) (IMP dehydrogenase 2) (IMPDH-II) (IMPD 2) IMPD2 OR IMPD2. 699 DHAC P00352 ↓ ↓ Aldehyde dehydrogenase, cytosolic 6.46 61,913 (EC 1.2.1.3) (class 1) (ALHDII) ALDH-E1 ALDH1 or ALDC 724 AMPL P00727 ↑ BOVINE Cytosol aminopeptidase (EC 6.32 57,846 3.4.11.1) (Leucine amino-peptidase) (LAP) ↑ See table 4 (Leucyl aminopeptidase) (proline aminopeptidase) (EC 3.4.11.5) (prolyl aminopeptidase) 724 DHAC P00352 ↑ Aldehyde dehydrogenase, cyto-solic 6.32 57,846 (EC 1.2.1.3) (class 1) (ALHDII) (ALDH-E1) 725 DHAC P00352 ↓ ↓ Aldehyde dehydrogenase, cyto-solic 6.30 56,853 (EC 1.2.1.3) (class 1) (ALHDII) (ALDH-E1) 760 SYW P23381 ↑ ↑ Tryptophanyl-tRNA synthetase (EC 5.78 52,099 6.1.1.2) (Tryptophan-tRNA ligase) 763 ↑ ↑ Novel unidentified protein 5.71 52,172 812 P60 P10809 ↓ ↓ Mitochondrial matrix protein P1 (P60 5.15 55,253 lymphocyte protein) 60 kD Chaperonin (heat shock protein 60) HSP-60 (protein CPN60) (GROEL) (HUCHA60) 826 YPH P19971 ↑ Thymidine phosphorylase (EC 5.14 50,432 2.4.2.4) (TDRPase) (platelet-derived endothelial cell growth factor) (PD- ECGF) (gliostatin) ECGF1 855 TBA1 P04687 ↑ Tubulin alpha-1 chain TUBA1 4.93 57,204 855 TBB1 P07437 ↑ Tubulin beta-1 chain 4.93 57,204 855 HSU5 Q99634 ↑ RIG-G 4.93 57,204 2513 864 TBB1 P07437 ↓ ↓ Tubulin beta-1 chain.TUBB1 4.76 54,251 864 TBB2 P05217 ↓ ↓ Tubulin beta-2 chain.TUBB2 4.76 54,251 864 PDI P07237 ↓ ↓ Protein disulfide isomerase (PDI) 4.76 54,251 (EC 5.3.4.1) Prolyl 4-hydroxylase beta subunit (EC 1.14.11.2) Cellular thyroid hormone binding protein (P55) 864 CBPH P16870 ↓ ↓ Carboxypeptidase H (EC 3.4.17.10) 4.76 54,251 (CPH) (Carboxypeptidase E) (CPE) (Enkephalin convertase) (prohormone processing carboxypeptidase) 909 ↓ ↓ Novel spectrum see table 6 4.41 64,739 963 IDHC P41562 ↑ RAT Isocitrate dehydrogenase 6.40 47,310 (NADP) cytoplasmic (EC 1.1.1.42) (Oxalo-succinate de-carboxylase) (IDH)(NADP+-spe-cific ICDH) (IDP) IDH1 See table 4 971 GATM P50440 ↓ Glycine amidinotransferase (EC 6.30 49,264 2.1.4.1) (L-arginine: glycine amidinotransferase) (transamidinase) AGAT 977 NUCM P17694 ↓ BOVINE NADH-Ubiquinone oxido- 6.36 45,388 reductase 49 kD subunit (EC 1.6.5.3)(EC 1.6.99.3) (Complex I 49 kD)(CI-49 kD) NDUFS2 See table 4 989 GATM P50440 ↓ Glycine amidinotransferase (EC 6.27 47,318 2.1.4.1) (L-arginine: glycine amidinotransferase) (transamidinase) AGAT 1001 SYW P23381 ↑ Tryptophanyl-tRNA synthetase (EC 5.93 45,147 6.1.1.2) (Tryptophan-tRNA ligase) 1012 SYW P23381 ↓ ↑ Tryptophanyl-tRNA synthetase (EC 5.82 45,316 6.1.1.2) (Tryptophan-tRNA ligase) 1012 PRS7 P35998 ↓ ↑ 26S Protease Regulatory Subunit 7 5.82 45,316 (MSS1 PROTEIN). 1013 ↑ Novel spectrum see table 6 5.79 44,636 1022 DHAG P49189 ↑ ↑ Aldehyde dehydrogenase, E3 5.71 44,824 isozyme (EC 1.2.1.3) (Gamma- aminobutyraldehyde dehydrogenase) (EC 1.2.1.19) (R-aminobutyraldehyde dehydrogenase). 1159 RINI P13489 ↑ Placental ribonuclease inhibitor 4.50 48,395 (Ribonuclease/an-giogenin inhibitor)(RAI)(RI) 1170 GR75 P38646 ↑ ↑ Mortalin, Mitochondrial stress-70 6.74 34,598 protein (75 KD Glu-cose Regulated Protein) GRP 75 1176 ↑ Novel unidentified protein 6.50 42,222 1186 ↑ Novel spectrum see table 6 6.46 36,234 1210 NED5 Q15019 ↓ NEDD5 protein homolog (KIAA0158) 6.14 40,411 NEDD5 or DIFF6) 1221 1A32 P10314 ↑ HLA class I histocompatibility 5.91 39,707 antigen, A-32(AW-19) alpha chain HLA-A OR HLAA 1241 ANX4 P09525 ↑ Annexin IV (Lipocortin IV) 5.64 38,609 (Endonexin I) (Chromobindin 4) (Protein II) (P32.5) (Placen-tal anticoagulant protein II) (PAP-II) (PP4-X) (35-beta Cal-cimedin) (Carbohydrate-binding protein P33/P41) 1241 DCUP P06132 ↑ Uroporphyrinogen decarboxylase (EC 5.64 38,609 4.1.1.37) (URO-D) UROD 1349 ANX2 P07355 ↓ Annexin II (Lipocortin II) (Calpactin 6.70 32,626 I heavy chain) (Chromobindin 8) (P36) (prtein I) (Placental anticoagulant protein IV) (PAP-IV) 1353 ↑ Novel spectrum see table 6 6.68 32,210 1368 ANX4 P09525 ↓ ↓ Annexin IV (Lipocortin IV) 6.36 30,916 (Endonexin I) (Chromobindin 4) (Protein II) (P32.5)(Placental anticoagulant protein II) (PAP-II) (PP4-X) (35-beta Calcimedin) (Carbohydrate-binding pro-tein P33/P41) 1376 G3P2 P04406 ↑ Glyceraldehyde 3-phosphate dehydrogenase, 6.19 34,534 Liver EC 1.2.1.12 1376 ANX2 P07355 ↑ Annexin II (Lipocortin II) (Calpactin 6.19 34,534 I heavy chain) (Chromobindin 8) (P36) (protein I) (Placental anticoagul-ant protein IV) (PAP-IV) ANX2 1400 ↓ Novel spectrum see table 6 5.77 33,725 1411 ANX4 P09525 ↑ Annexin IV (Lipocortin IV) 5.49 31,904 (Endonexin I) (Chromobindin 4) (Protein II) (P32.5) (Placen-tal anticoagulant protein II) (PAP-II) (PP4-X) (35-beta cal-cimedin) (Carbohydrate-binding protein P33/P41) 1421 ANX2 P07355 ↑ Annexin II (Lipocortin II) (Calpactin 5.43 33,800 I heavy chain) (Chromobindin 8) (P36) (protein I) (Placental anticoagulant protein IV) (PAP-IV) 1421 IPYR P37980 ↑ BOVINE Inorganic pyrophosphatase 5.43 33,800 (EC 3.6.1.1) (Pyrophosphate phospho-hydrolase) (PPASE) PP See table 4 1426 ANX2 P07355 ↑ Annexin II (Lipocortin II) (Calpactin 5.28 33,836 I heavy chain) (Chromobindin 8) (P36) (protein I) (Placental anticoagulant protein IV) (PAP-IV) 1477 ↑ Novel spectrum see table 6 4.48 35,393 1531 IGUP Q06323 ↑ ↑ Interferon gamma up-regulated I- 5.68 28,980 5111 protein (IGUP I-5111) 1545 CAPB P47756 ↑ F-Actin capping protein beta subunit 5.36 28,940 (CAPZ) CAPZB. 1545 CATD P07339 ↑ Cathepsin D (EC 3.4.23.5) CTSD 5.36 28,940 1545 PRC2 P25786 ↑ Proteasome component C2 (EC 5.36 28,940 3.4.99.46) (Macropain subunit C2) (Proteasome NU chain) (multicatalytic endopeptidase complex subunit C2) (30 kD prosomal protein) (PROS-30) PSMA1 or PSC2 or PROS30 1549 ↑ Novel spectrum see table 6 5.40 28,949 1609 SODM P04179 ↑ ↑ Superoxide dismutase (MN) (EC 6.83 21,212 1.15.1.1) SOD2 1629 ↑ Novel spectrum see table 6 6.15 25,165 1632 TPIS P00938 ↑ Triosephosphate isomerase (EC 6.11 27,492 5.3.1.1) (TIM) TPI1 OR TPI 1632 ULA6 P30041 ↑ 24 kD protein 6.11 27,492 1632 PRCF P40306 ↑ Proteasome component MECL-1 (EC 6.11 27,492 3.4.99.46) (Macropain sub-unit MECL-1) (multicatalytic endopeptidase complex subunit MECL-1) PSMB10 OR MECL1 1664 S85655 P35232 ↓ Prohibitin PHB 5.44 27,014 1664 HS27 P04792 ↓ Heat shock 27 kD protein (HSP 27) 5.44 27,014 (Stress-responsive protein 27)(SRP27)(Estrogen-regulated 24 kD protein) (28 kD Heat shock protein) HSPB1 OR HSP27 1685 ↑ Novel spectrum see table 6 4.81 23,690 1689 ↓ Novel spectrum see table 6 4.69 22,974 1697 HMG1 P09429 ↑ ↑ High mobility group protein HMG1 4.60 24,122 (HMG-1) 1707 ↑ ↑ Novel spectrum see table 6 6.50 24,673 1715 ↓ ↓ Novel spectrum see table 6 5.82 26,369 1737 PRC7 P28065 ↑ Proteasome chain 7 (EC 3.4.99.46) 4.56 19,832 (Macropain chain 7) (Multicatalytic endopeptidase complex chain 7) (ring12 pro-tein) PSMB9 or LMP2 or RING12 1766 ↓ ↓ Novel spectrum see table 6 5.61 17,650 1800 ↓ Novel spectrum see table 6 4.47 17,697 1853 B2MG P01884 ↑ ↑ Beta-2-microglobulin 5.73 7,077 1861 G267406 ↓ 6.28 60,877 1902 ↑ Novel spectrum see table 6 5.52 71,993 1923 AMPL P00727 ↑ BOVINE Cytosol aminopeptidase (EC 6.45 59,804 3.4.11.1) (Leucine aminopeptidase) (LAP) (Leucyl aminopeptidase) (proline aminopeptidase) (EC 3.4.11.5) (prolyl aminopeptidase) See table 4 1923 DHAC P00352 ↑ Aldehyde dehydrogenase, cyto-solic 6.45 59,804 (EC 1.2.1.3) (class 1) (ALHDII) (ALDH-E1) 1923 DHE3 P00367 ↑ Glutamate dehydrogenase 1 (EC 6.45 59,804 1.4.1.3) (GDH) 1930 ↓ ↓ Novel unidentified protein 4.73 47,472 1935 ↑ ↑ Novel spectrum see table 6 6.51 49,146 1996 ENPL P14625 ↓ Endoplasmin (94 kD Glucose- 4.92 90,497 regulated protein) (grp94) (gp96 homolog) (Tumour rejec-tion antigen 1) TRA1 2036 AMYS P04745 ↑ alpha-Amylase salivary (EC 3.2.1.1) 6.44 70,123 (1,4-alpha-D-Glucan glucanohydrolase) AMY1A OR AMY1 672 DCE2 Q05329 ↑ ↑ Glutamate decarboxylase 65 kD 4.46 78,008 isoform 2041 ↑ ↑ Novel spectrum see table 6 6.35 68,704 2079 ↓ ↓ Novel spectrum see table 6 5.86 47,814 2104 ILEU P30740 ↑ Leukocyte elastase inhibitor (LEI) 5.37 41,459 (monocyte/neutrophil elastase inhibitor) (EI) ELANH2 or PI2 2104 1B08 P30463 ↑ HLA class I histocompatibility 5.37 41,459 antigen, BW-65(B-14) B*1402 alpha chain HLA-B or HLAB 2145 LITA P05451 ↑ (Islet cells regeneration 5.38 13,733 factor)(ICRF) Lithostathine 1 alpha (Pancreatic stone protein) (PSP) Pancreatic thread protein) (PTP) 2159 ATPA P25705 ↑ ↑ ATP Synthase alpha chain, 6.83 64,414 mitochondrial (EC 3.6.1.34) ATP5A1. 2276 ER60 P30101 ↑ Protein disulfide isomerase ER-60 5.68 52,329 (EC 5.3.4.1) (ERP60) (58 KD Microsomal protein) (P58) (GRP58) (ERP57) 2276 SYW P23381 ↑ Tryptophanyl-tRNA synthetase (EC 5.68 52,329 6.1.1.2) (Tryptophan-tRNA ligase) 2286 SYW P23381 ↑ Tryptophanyl-tRNA synthetase (EC 5.64 52,988 6.1.1.2) (Tryptophan-tRNA ligase) 2286 ER60 P30101 ↑ protein)(P58)(GRP58)(ERP57) 5.64 52,988 2354 ↓ Novel spectrum see table 6 5.75 44,151 2368 ILEU P30740 ↑ Leukocyte elastase inhibitor (LEI) 5.27 41,658 (monocyte/neutrophil elastase inhibitor) (EI) ELANH2 or PI2 2368 1B39 P30480 ↑ HLA Class I histocompatibility 5.27 41,658 antigen, BW-42 B*4201 alpha chain HLA-B or HLAB 2382 ↓ ↓ Novel spectrum see table 6 5.74 36,688 2408 ↑ Novel spectrum see table 6 5.74 29,142 2411 ↑ ↑ Novel spectrum see table 6 7.17 21,901 2422 ↑ ↑ Novel unidentified protein 4.71 288,000 2423 ↑ ↑ Novel unidentified protein 4.65 286,000 13298 SYW P23381 ↑ Tryptophanyl-tRNA synthetase (EC 5.86 52,288 6.1.1.2) (Tryptophan-tRNA ligase) 13652 ↑ Novel spectrum see table 6 4.98 35,726 14098 ↑ Novel spectrum see table 6 6.83 21,691 14103 SODM P04179 ↑ ↑ Superoxide dismutase (MN) (EC 7.41 20,628 1.15.1.1) SOD2

TABLE 2 HUMAN ISLET CELL PROTEINS CHARACTERISED BY MS. IDENTIFIED, IDENTIFIED RELATING TO ANOTHER SPECIES AND UNIDENTIFIED PROTEINS. Gene IL1β Cytokin Protein Obs. Mol. Spot # Name Identifier 1500 Mix Name pI Wt 007 HBP Q00341 ↓ High density lipoprotein binding 7.22 96,450 protein (HDL-binding protein) 026 ↑ Novel spectrum see table 7 8.20 79,860 035 ↑ ↑ Novel spectrum see table 7 7.68 79,138 060 ↓ ↓ Novel spectrum see table 7 8.34 72,134 076 ↑ Novel spectrum see table 7 7.82 68,538 085 ↑ Novel spectrum see table 7 7.52 68,772 128 ↑ Novel spectrum see table 7 8.64 63,052 130 ↑ Novel spectrum see table 7 8.43 64,647 140 KPY1 P14618 ↑ Pyruvate kinase, M1 (muscle 8.31 58,557 isozyme) (EC 2.7.1.40) 142 ROM P52272 ↓ Heterogeneous nuclear ribonucleoprotein 8.21 62,958 M (HNRNP M) 147 ROM P52272 ↓ ↓ Heterogeneous nuclear ribonucleoprotein 8.03 62,953 M (HNRNP M) 147 CPT2 P23786 ↓ ↓ Mitochondrial carnitine palmitoyltransferase 8.03 62,953 II (EC 2.3.1.21) 157 PUT2 P30038 ↓ ↓ Delta-1-pyrroline-5-carboxylate 7.55 60,341 dehydrogenase(EC 1.5.1.12) 157 CATA P04040 ↓ ↓ Catalase (EC 1.11.1.6) 7.55 60,341 167 Q05329 ↑ ↑ Glutamate decarboxylase 65 kD 7.30 61,344 isoform 171 ↓ Novel spectrum see table 7 7.10 61,058 187 ↑ Novel spectrum see table 7 9.21 51,881 188 ↑ Novel spectrum see table 7 9.17 51,844 195 ↓ Novel spectrum see table 7 9.07 56,351 241 ATPA P25705 ↓ ↓ ATP Synthase alpha chain, 7.54 53,358 mitochondrial (EC 3.6.1.34) 243 ↑ Novel spectrum see table 7 7.55 55,588 247 DHE3 P00367 ↑ Glutamate dehydrogenase 1 7.46 55,349 (EC 1.4.1.3) 247 PBEF P43490 ↑ Pre-B cell enhancing factor 7.46 55,349 256 DHE3 P00367 ↑ Glutamate dehydrogenase 1 6.90 57,118 (EC 1.4.1.3) 256 G6PD P11413 ↑ Glucose-6-phosphate 1-dehydrogenase 6.90 57,118 (EC 1.1.1.49) 263 ↓ ↓ Novel unidentified protein 9,619 49,828 270 ↓ Novel spectrum see table 7 9.14 47,719 308 GABT P80404 ↓ 4-Aminobutyrate aminotransferase, 7.96 51,251 mitochondrial (EC 2.6.1.19) See table 5 339 ODPA P08559 ↑ Pyruvate dehydrogenase E1 6.95 49,851 component, alpha subunit, somatic (EC 1.2.4.1) 339 TAP-A Y13582 ↑ Transporter associated with 6.95 49,851 antigen processing subunit TAP-A 339 IDHP P48735 ↑ Isocitrate dehydrogenase 6.95 49,851 (NADP), mitochondrial (EC 1.1.1.42) See table 5 369 RAB7 P51149 ↑ RAS-Related protein RAB-7 8.48 42,835 369 ALFA P04075 ↑ Fructose-bisphosphate aldolase 8.48 42,835 A (muscle)(EC 4.1.2.13) 376 DBDD Q04828 ↑ Trans-1,2-dihydrobenzene-1,2- 8.14 41,509 diol dehydrogenase (EC 1.3.1.20) 376 ALFA P04075 ↑ Fructose-bisphosphate aldolase 8.14 41,509 A (muscle)(EC 4.1.2.13) 377 GAPDHG J04038 ↑ Novel unidentified protein 8.23 39,510 386 THIL P24752 ↓ Acetyl-CoA acetyltransferase, 7.82 44,584 mitochondrial (EC 2.3.1.9) 386 PGK1 P00558 ↓ Phosphoglycerate kinase 1 (EC 7.82 44,584 2.7.2.3) 418 MDHP G290614 ↓ Malate dehydrogenase See 8.89 35,367 table 5 418 ROA1 P09651 ↓ Heterogeneous nuclear ribonucleoprotein 8.89 35,367 A1 (Helix- destabilizing protein)(Single- strand binding protein) (HNRNP core protein A1) 421 ↓ Novel spectrum see table 7 8.95 32,958 449 ↑ Novel spectrum see table 7 7.85 37,503 453 POR2 P45880 ↓ Voltage-dependent anion-selective 7.73 35,149 channel protein 2 (VDAC2) (Outer mitochondrial membrane protein porin) 460 NC5R P00387 ↓ ↑ NADH-cytochrome B5 reductase 7.41 36,378 (EC 1.6.2.2) 464 MDHC P40925 ↓ Malate dehydrogenase, cytoplasmic 7.01 36,742 (EC 1.1.1.37) 474 E123123 E123123 ↓ ↓ NIPSNAP1 protein [2769648] 9.44 25,090 See table 5 500 ↑ Novel unidentified protein 7.85 30,805 508 ↑ Novel spectrum see table 7 7.71 26,937 509 ↑ Novel spectrum see table 7 7.32 29,812 515 PMGB P18669 ↓ Phosphoglycerate mutase, brain 6.86 25,560 (EC 5.4.2.1) 517 ↑ Novel spectrum see table 7 9.79 20,443 519 NGAL P80188 ↑ Alpha-2-microglobulin-related 9.55 20,534 subunit of MMP-9 (Lipocalin-2) 532 ↑ ↑ Novel spectrum see table 7 8.20 26,749 536 ETFB P38117 ↑ Electron transfer flavoprotein 8.11 26,840 beta-subunit (beta-ETF) See table 5 536 O14818 O14818 ↑ Proteasome subunit XAPC7 8.11 26,840 538 TDX2 Q06830 ↑ Natural killer cell enhancing 8.02 21,570 factor A 538 COF1 P23528 ↑ Cofilin, non-muscle isoform. 8.02 21,570 538 LEG3 P17931 ↑ Galectin-3 (Galactose-specific 8.02 21,570 lectin 3) (MAC-2 antigen)(IGE- binding protein) (35 kd lectin) 538 SODM P04179 ↑ Superoxide dismutase (MN) 8.02 21,570 (EC 1.15.1.1) 544 ES1 P30042 ↓ ES1 Protein homolog (protein 7.74 23,665 KNP-I) (GT335) 546 SODM P04179 ↑ ↑ Superoxide dismutase (MN) 7.74 21,122 (EC 1.15.1.1) 549 HSSOD X07834 ↑ ↑ Superoxide dismutase (MN) (EC 7.63 20,690 1.15.1.1) 551 SODM P04179 ↑ ↑ Superoxide dismutase (MN) (EC 7.45 20,108 1.15.1.1) 558 ↓ Novel spectrum see table 7 10.01 16,701 559 ↓ ↓ Novel spectrum see table 7 10.06 16,640 560 ↓ ↓ Novel spectrum see table 7 9.43 18,700 565 SM22 Q01995 ↓ ↓ Smooth muscle protein 22- 8.86 21,594 alpha (SM22-alpha) (Transgelin) 565 PRCE P28074 ↓ ↓ Proteasome epsilon chain (EC 8.86 21,594 3.4.99.46) 568 TDX2 Q06830 ↑ Natural killer cell enhancing 8.58 22,493 factor A 572 SM2H P37802 ↑ SM22-alpha homolog (HA1756) 8.21 19,370 577 SODM P04179 ↑ ↑ Superoxide dismutase (MN) 7.75 19,871 (EC 1.15.1.1). 580 ↑ Novel unidentified protein 10.30 14,098 596 COF1 P23528 ↓ ↓ Cofilin, non-muscle isoform 8.36 16,170 605 CYPH P05092 ↓ ↓ Peptidyl-prolyl cis-trans 7.94 27,866 isomerase A (EC 5.2.1.8) 609 ↓ ↓ Novel spectrum see table 7 8.79 9,751 635 Q13122 Q13122 ↓ 100 kDa coactivator See table 5 7.54 83,777 642 GATM P50440 ↓ Glycine amidinotransferase (EC 7.15 51,281 2.1.4.1)(L-arginine: glycine amidinotransferase) 642 ENOA P06733 ↓ Alpha enolase (EC 4.2.1.11) 7.15 51,281 656 TDX2 Q06830 ↑ Thioredoxin peroxidase 2 8.80 37,605 (Thioredoxin-dependent peroxide reductase 2) (Prolifera-tion- associated protein PAG) (Natural killer cell enhancing factor A) 693 ALFA P04075 ↓ ↓ Fructose-bisphosphate aldolase 9.65 49,829 A (muscle) (EC 4.1.2.13) 702 C10H Q16181 ↑ CDC10 Protein homolog 9.15 46,065 719 ↑ ↑ Novel spectrum see table 7 8.43 25,936 720 PRCY P28062 ↑ ↑ Proteasome component C13 8.26 22,960 (EC 3.4.99.46) 720 TDX2 Q06830 ↑ ↑ Thioredoxin peroxidase 2 8.26 22,960 (Thioredoxin-dependent peroxide reductase 2) (Prolifera-tion- associated protein PAG) (Natural killer cell enhancing factor A) 729 ↓ Novel spectrum see table 7 7.58 128,532 780 G3P2 P04406 ↑ Glyceraldehyde 3-phosphate 8.02 39,330 dehydrogenase, liver (EC 1.2.1.12) 780 ROA2 P22626 ↑ Heterogeneous nuclear ribonucleoproteins 8.02 39,330 A2/B1 (HNRNP A2 and HNRNP B1). 786 ANX2 P07355 ↓ Annexin II (Lipocortin II) 7.09 38,665 811 ↑ Novel unidentified protein 8.38 23,437 816 PRCE P28074 ↓ Proteasome epsilon chain (EC 8.97 21,177 3.4.99.46) 825 HSU889$$ O00586 ↑ hm45 9.58 15,624 828 NDKB P22392 ↓ Nucleoside diphosphate kinase 8.70 15,409 B (EC 2.7.4.6) 829 ↓ Novel spectrum see table 7 8.59 15,630 834 CYPB P23284 ↓ ↓ Peptidyl-prolyl cis-trans 9.64 13,601 isomerase B (EC 5.2.1.8) 836 ↓ ↑ Novel spectrum see table 7 8.19 20,638 837 ↑ Novel spectrum see table 7 8.17 21,390 3755 ↓ ↓ Novel unidentified protein 10.56 12,875 3879 ↓ Novel spectrum see table 7 8.35 16,365 3889 FLRE P30043 ↑ ↑ Flavin reductase (EC 1.6.99.1) 7.93 22,328 4325 PRS8 P47210 ↓ 26S Protease regulatory subunit 7.47 48,652 8 (Proteasome subunit P45) (Thyroid hormone receptor interacting protein 1) (TRIP1) 6600 ↑ Novel spectrum see table 7 9.04 21,431

TABLE 3 PROTEINS DETECTED IN HUMAN ISLET CELL MEDIA AND CHARACTERISED BY MS IDENTIFIED AND UNIDENTIFIED PROTEINS. Gene IL1β Cytokin Protein Spot # Name Identifier 1500 Mix Name Obs. pI Mol.Wt 63SPI ALBU P02768 ↑ ↑ Serum albumin ALB 4.74 64,376 83SPI ALBU P02768 ↑ ↑ Serum albumin ALB 4.94 58,998 122SPI ↑ See table 8 7.36 34,564 123SPI ↓ ↓ See table 8 6.65 36,736 126SPI ↓ ↓ See table 8 5.42 35,578 130SPI ↑ See table 8 4.65 32,480 135SPI ↓ See table 8 6.36 30,916 140SPI ↓ ↓ See table 8 4.73 29,274 160SPI ↓ See table 8 5.25 21,101 168SPI ALBU P02768 ↓ ↓ Serum albumin ALB 5.27 18,521 213SPI ALBU P02768 ↑ Serum albumin ALB 5.88 31,124 215SPI ALBU P02768 ↓ Serum albumin ALB 4.98 12,7138 215SPI TRFE P02787 ↓ Serotransferrin (Siderophilin) 4.98 12,7138 (beta-1-Metal binding globulin) TF 218SPI See table 8 6.27 632,16 248SPI ↓ ↓ See table 8 4.67 292,24 258SPI ALBU P02768 ↑ Serum albumin ALB 6.15 246,493 258SPI HSIG Y14737 ↑ Immunoglobulin lambda 6.15 246,493 G1LH heavy chain 277SPI ↓ ↓ See table 8 5.05 15,752 304SPI ↓ ↓ See table 8 6.71 36,953 314SPI ↑ See table 8 6.46 56,100 320SPI ALBU P02768 ↓ Serum albumin ALB 5.89 9,210 338SPI ↓ ↓ See table 8 4.67 16,603 1157SPI ALBU P02768 ↓ ↓ Serum albumin ALB 5.47 18,501 1157SPI HSIG Y14737 ↓ ↓ Immunoglobulin lambda 5.47 18,501 G1LH heavy chain

TABLE 4 HUMAN ISLET CELL PROTEINS CHARACTERISED BY MS The peptide molecular weight values are read directly out of the mass spectrometer and are thus associated with the accuracies normally obtained with such instrumentation. These mass values are from human islet cell proteins recovered from the IEF gels and have been identified by reference to proteins from other species, recorded in publicly available databases (whether nucleotide or protein sequence). IEF spot 724: 984.4406 1004.6172 1045.5750 1084.6248 1137.5220 1172.5513 1181.5787 1188.5267 1189.5705 1193.5450 1209.5364 1232.6502 1237.5782 1244.6390 1273.6111 1365.6163 1381.6246 1418.7151 1434.7000 1437.7885 1476.6972 1525.7884 1544.7837 1583.7063 1589.8061 1601.7708 1618.7619 1630.8195 1645.8241 1684.8527 1700.7857 1728.8839 1730.8908 1745.9084 1781.8147 1794.8168 1826.8641 1860.9918 1918.9139 1940.9287 1989.9645 2005.9335 2055.9358 2077.1362 2095.9846 2211.0999 2328.1808 2674.4376 3198.8492 IEF spot 963: 903.5003 976.5949 1045.5749 1064.5752 1168.5158 1198.6904 1319.6604 1341.6072 1350.6473 1379.6844 1437.8828 1445.7037 1501.7661 1509.6583 1515.7456 1596.7730 1707.7736 1790.9116 1794.8513 1797.8409 1808.9138 1838.9222 1852.9407 1878.9424 1946.9897 2122.0285 2150.0260 2211.0999 2406.2399 2559.1687 2585.3182 IEF spot 977: 842.5100 973.4918 1021.4022 1031.4738 1037.3794 1045.4938 1060.7676 1075.5956 1151.6148 1159.5482 1179.5398 1196.5457 1263.5613 1267.6112 1277.6170 1279.5629 1307.5846 1320.5246 1329.5717 1386.6334 1437.7641 1458.6907 1472.6424 1475.6892 1536.8279 1597.8518 1674.8387 1700.9337 1717.7814 1794.8163 1802.9448 1826.7513 1873.0013 1881.0435 1888.9044 1942.0398 1994.0735 2211.0999 2225.1090 2231.1803 2455.2958 IEF spot 1421: 957.5447 1053.4829 1075.5106 1114.5114 1146.5867 1151.6223 1163.5530 1177.5601 1197.6195 1222.5030 1227.5776 1277.6330 1291.6297 1302.6520 1307.6147 1317.6238 1327.6099 1343.6147 1383.6003 1391.6233 1393.6514 1396.7482 1403.6989 1421.6244 1427.8312 1434.6764 1439.7253 1460.6249 1469.7214 1475.6805 1477.6583 1487.7037 1489.6919 1515.7063 1540.7410 1542.7700 1544.7626 1565.7587 1570.7737 1588.7486 1613.8336 1619.8079 1630.8408 1639.7890 1645.7757 1650.8568 1674.8047 1678.8083 1685.7486 1694.8735 1701.7636 1726.8556 1741.8646 1749.8863 1763.7788 1789.9034 1805.8828 1837.9159 1850.9476 1863.9118 1869.9458 1923.9277 1938.8968 1988.0785 1993.9638 2052.0445 2101.0154 2139.0677 2155.0785 2175.8964 2196.1696 2211.1000 2225.1153 2230.0816 2444.2075 2461.1385 IEF spot 1861: 986.6183 1099.5910 1277.6498 1305.6530 1383.6670 1390.5687 1462.7108 1475.7014 1489.7417 1516.7727 1544.7517 1555.7811 1590.7901 1594.8311 1638.7729 1644.8520 1654.8019 1661.7714 1701.8654 1707.7328 1716.8788 1746.9048 1756.9955 1794.8406 1799.9569 1802.8989 1837.9500 1851.9245 1900.9804 1920.1066 1990.9681 1993.9618 2062.0419 2211.1082 2225.1014 2272.1417 2298.1959 2472.2712 2502.2031 IEF spot 1923: 1004.6256 1045.5750 1084.6033 1137.4897 1172.5717 1181.5737 1188.5163 1189.5777 1193.5510 1209.5453 1232.6509 1237.5933 1263.6638 1273.6130 1277.6849 1307.6799 1365.6431 1381.6188 1394.7306 1418.7243 1425.6424 1437.8278 1444.6997 1455.7280 1475.7475 1525.7932 1544.7815 1583.7130 1589.7876 1601.7387 1618.7568 1630.8242 1645.8190 1669.8494 1684.8860 1686.8789 1700.8011 1707.8472 1728.8715 1737.8908 1746.8984 1748.9094 1779.9697 1782.8774 1794.8152 1835.9384 1838.9265 1860.9665 1908.0260 1915.9525 1920.9264 1940.9263 1989.9395 2055.9253 2077.1037 2095.9638 2211.0999 2278.2563 2328.1495 3197.5962 3494.9900

TABLE 5 HUMAN ISLET CELL PROTEINS CHARACTERISED BY MS The peptide molecular weight values are read directly out of the mass spectrometer and are thus associated with the accuracies normally obtained with such instrumentation. These mass values are from human islet cell proteins recovered from the NEPHGE gels and have been identified by reference to proteins from other species, recorded in publically available databases (whether nucleotide or protein sequence). NEPHGE spot 308: 1016.5065 1032.4876 1035.6038 1055.5031 1148.5696 1179.6000 1197.6175 1211.5699 1221.5578 1229.7271 1264.6730 1265.6893 1277.6546 1283.7385 1307.6327 1317.7154 1338.7857 1352.8162 1379.7060 1383.6587 1405.6557 1434.7509 1475.7249 1487.7638 1505.7494 1527.6946 1539.7651 1549.7666 1561.7328 1594.8652 1612.7713 1628.7452 1631.7017 1636.6749 1638.8614 1645.8028 1707.7680 1710.8218 1719.7991 1757.9016 1770.9237 1786.9209 1794.8343 1812.9308 1822.9402 1837.9966 1852.9594 1940.9424 1988.0063 1993.9935 2064.1358 2149.0169 2155.2298 2196.1764 2211.0999 2250.1119 2596.2598 2674.3040 3150.8016 NEPHGE spot 339: 1009.3940 1171.5822 1179.5496 1226.4567 1277.6437 1320.6468 1383.6308 1405.4537 1411.7060 1426.7141 1437.8630 1471.7116 1475.7304 1483.6959 1493.6772 1501.7870 1542.8788 1592.6512 1596.7767 1638.8489 1695.8217 1707.7969 1715.8272 1778.8540 1794.8041 1797.8152 1819.8835 1823.9229 1838.9469 1851.9609 1878.9472 1940.9953 1962.8823 1978.9525 1993.9931 2018.0026 2211.1201 2225.1558 2274.1434 2283.1658 2299.2521 2344.3761 2383.9712 2402.2620 2406.3035 2431.0818 2705.2697 2719.1012 2731.3472 2807.4831 NEPHGE spot 418: 822.4000 832.4732 842.5100 908.6270 928.4098 992.4582 1033.4602 1045.4933 1065.4224 1073.4967 1107.4692 1116.4852 1147.5546 1157.4959 1165.4439 1167.4800 1179.5001 1201.5553 1218.5409 1232.5022 1277.6204 1299.5559 1307.5788 1352.6447 1357.6185 1384.6983 1393.6325 1406.5687 1422.5837 1427.6627 1437.6719 1454.6286 1470.6347 1475.6829 1493.6574 1500.6009 1503.6764 1560.7424 1628.7381 1657.7530 1694.6866 1699.7547 1707.7213 1716.8199 1741.7043 1751.7916 1784.8936 1791.7001 1838.8855 1851.8301 1867.8840 1987.9418 2211.0999 2365.2112 2383.9402 NEPHGE spot 474: 679.5296 706.4771 714.4944 730.4229 991.5260 1012.5126 1092.5579 1108.5016 1212.5737 1225.5337 1277.6968 1307.6588 1446.8720 1475.7748 1604.7245 1710.9075 1772.9576 1788.9636 1794.8920 1867.0268 2018.1137 2174.2120 2211.2344 2401.4191 2789.6777 NEPHGE spot 536: 853.5924 945.5755 978.5561 998.5303 1011.6255 1045.5785 1054.6595 1066.5559 1102.5904 1179.6000 1183.7368 1184.6987 1277.6972 1291.7293 1295.7841 1304.7071 1307.7127 1339.7158 1403.7144 1617.8575 1634.8604 1664.8766 1666.7879 1683.9517 1737.8633 1794.8280 1801.8590 1812.0373 1902.8723 1907.8546 1918.9088 1940.9273 1945.9582 1993.9928 2026.0434 2065.0661 2196.0354 2211.0999 2449.2132 2577.3710 NEPHGE spot 635, 842.5456 1011.6439 1036.5530 1045.5684 1048.6357 1064.5920 1071.5091 1082.6031 1092.5681 1106.6437 1157.5750 1179.6000 1232.6820 1277.7070 1307.6714 1314.7441 1320.5891 1323.6417 1338.7554 1350.6851 1357.6931 1383.6509 1389.6969 1396.6928 1401.6416 1407.7207 1429.6767 1434.7677 1461.6809 1475.7583 1493.7287 1535.8219 1620.8873 1638.8669 1657.7833 1665.9997 1702.8599 1707.7849 1709.8196 1716.8613 1724.9400 1757.9150 1794.8189 1829.8908 1838.9142 1851.9404 1867.9269 1940.9311 1959.9202 1993.9642 2047.0784 2150.0418 2211.0999

TABLE 6 HUMAN ISLET CELL PROTEINS CHARACTERISED BY MS The peptide molecular weight values are read directly out of the mass spectrometer and are thus associated with the accuracies normally obtained with such instrumentation. These mass values are from human islet cell proteins recovered from the IEF gels and do not match any protein recorded in publicly available databases (whether nucleotide or protein sequence). IEF spot 8: 1045.6833 1179.6990 1277.8454 1475.8751 1794.9853 1816.9418 1833.9383 2211.3200 2225.3308 2231.4079 2239.3200 2249.2374 2298.4052 2721.5682 2807.6137 3340.0959 IEF spot 370: 842.5100 995.5638 1009.5700 1023.5915 1045.4798 1155.5365 1254.5757 1264.5343 1275.5888 1348.5604 1419.6498 1475.6669 1794.7643 1940.9135 1950.0052 1993.9562 2211.0999 2403.2608 IEF spot 473: 1008.2678 1094.4059 1151.4450 1208.4057 1254.4806 1267.4619 1277.4823 1285.4220 1315.4963 1355.4978 1375.5110 1383.4741 1475.5626 1488.6075 1497.5317 1506.5091 1555.6522 1589.7591 1607.6268 1615.6441 1624.6694 1668.7791 1697.7066 1700.8164 1706.8476 1716.7516 1761.7701 1794.7184 1826.7320 1838.8344 1853.8072 1880.9486 1919.9092 1922.8662 1940.8495 1993.9770 2034.9080 2107.0836 2211.0999 2225.1068 2231.2066 IEF spot 524: 842.5100 1045.5152 1201.6168 1404.6128 1481.7153 1561.8987 1794.7933 1826.8585 1940.9415 2211.0999 2225.1233 2230.1675 2240.1371 2284.2128 2298.2138 2315.1913 IEF spot 535: 1061.3738 1077.1038 1277.6330 1352.6221 1398.7634 1470.7513 1561.2142 1561.6867 1637.8923 1670.3631 1674.8225 1794.8100 1826.8587 1838.9166 1852.9038 1940.9301 1942.9249 1993.9472 2027.0373 2083.0422 2085.0098 2914.5719 IEF spot 551: 973.6111 1036.5433 1109.4879 1179.6000 1211.6334 1263.7032 1277.7166 1308.6403 1352.5835 1365.6650 1369.6804 1383.6823 1434.7299 1475.7586 1487.7383 1493.7134 1637.9332 1687.8217 1699.8350 1707.7806 1716.8403 1784.7226 1794.8425 1796.9945 1838.0168 1993.9891 2211.1000 2297.1887 2663.2473 2705.1573 2720.2757 2807.3089 3121.4704 IEF spot 651: 842.5100 1263.6624 1277.6589 1332.6843 1404.7025 1419.6938 1491.7324 1508.9459 1512.7036 1517.8358 1587.8030 1638.8522 1729.9886 1749.0137 1794.8426 1838.9400 1844.0229 1851.9611 1922.9308 1934.0288 1940.9595 1987.0910 1993.9490 2000.0800 2040.1006 2043.1000 2083.9484 2093.0841 2124.0855 2137.1187 2165.0434 2211.1000 2225.1111 2231.1652 2284.1895 2298.2321 IEF spot 656: 842.5100 1045.5397 1176.4935 1179.5345 1277.6399 1307.6511 1320.5211 1323.6361 1354.6520 1357.6312 1383.5951 1433.7019 1450.6642 1461.6939 1473.6322 1475.7114 1498.5909 1561.6855 1613.7589 1707.7832 1716.8349 1718.8505 1794.7881 1838.9436 1854.6243 1882.0321 1908.9185 1992.0052 1993.9823 2130.2472 2211.0999 2225.0997 2284.1469 2298.1604 IEF spot 909: 842.5100 1045.5307 1066.0095 1077.1195 1140.5949 1151.6268 1173.5611 1179.5166 1234.5855 1263.6313 1277.6287 1301.6433 1307.6049 1314.6572 1320.5066 1357.6246 1383.5860 1434.6890 1475.6862 1493.6750 1532.7323 1535.8087 1547.6337 1623.8526 1638.7999 1669.8425 1707.7065 1716.8138 1739.9255 1766.8347 1791.7279 1794.7902 1806.8742 1838.9270 1852.9250 1868.9303 1881.0355 1890.9128 1940.9064 1944.9914 1993.9906 2111.9138 2171.1384 2211.1000 2225.1044 2230.1917 2240.1803 2298.1557 2384.9693 IEF spot 1013: 842.5100 918.2719 995.6119 1045.5322 1050.5029 1144.5461 1191.6716 1201.6286 1214.5566 1308.4854 1319.6821 1341.5464 1474.7240 1536.8891 1604.7969 1701.9124 1707.8899 1781.8221 1794.8021 1801.0348 1826.8456 1916.8642 1923.9753 1940.9620 1982.9495 1999.9878 2083.9889 2112.0200 2211.0999 2225.1149 2314.1955 IEF spot 1186: 842.5100 1277.6348 1795.8217 1918.9837 2211.1000 IEF spot 1353: 842.5100 870.5290 944.4994 981.5298 989.5240 995.5611 998.5569 1009.5790 1023.5824 1035.4838 1045.5007 1111.4667 1172.5993 1179.5128 1198.5672 1240.5947 1263.5655 1277.6049 1307.5663 1314.6804 1411.6442 1421.5773 1427.7229 1460.6109 1475.6725 1533.7745 1542.7685 1544.7350 1614.8168 1699.7857 1707.7142 1716.8069 1763.7734 1794.7732 1940.9113 1993.9414 2082.9974 2211.0999 2284.1656 2298.1924 2315.1733 2346.2393 2402.2586 IEF spot 1400: 1077.4235 1545.1665 1557.1800 1615.2899 1629.3171 1639.2424 1644.2703 1650.2177 1679.2655 1764.2349 1781.2559 1795.2691 1827.3200 1923.3377 1941.4215 2012.4217 2084.4777 2196.6995 2211.6313 2298.7533 2314.7422 IEF spot 1477: 842.5100 1045.5856 1061.4107 1201.6810 1243.6338 1298.7593 1314.7386 1353.7389 1442.6934 1794.8225 1816.8090 1826.8278 1940.9308 1962.9235 2083.9815 2211.0999 2225.0952 2233.0880 2239.1110 2247.1584 2259.1687 2283.1243 2298.1830 2300.1578 2300.1578 2314.2315 842.5100 IEF spot 1549: 944.5832 952.5302 963.4854 968.5148 989.6031 1011.4641 1038.5338 1045.5750 1085.6121 1109.5172 1123.4616 1141.4897 1151.5241 1169.4984 1180.5681 1239.5897 1254.5633 1263.6304 1293.6086 1332.6938 1404.7182 1449.7184 1453.7806 1462.6382 1481.7035 1487.7197 1504.7506 1534.7408 1550.6930 1601.8160 1620.7593 1696.8256 1776.8086 1794.8104 1834.9544 2082.9407 2104.9489 2120.9161 2191.1179 2211.0999 2307.1286 3494.7356 IEF spot 1629: 842.5100 1081.5213 1163.5498 1277.6486 1510.6347 1602.8306 1614.7873 1622.7896 1766.8741 1783.9029 1794.8229 1821.9804 1889.0158 1905.9656 1940.9499 1994.9698 2001.0446 2015.9908 2100.9779 2194.1209 2206.0567 2211.1000 2222.1179 2227.0998 2232.1773 2239.1005 2284.1852 2298.1724 2315.1713 2402.2609 IEF spot 1685: 842.5100 1022.5918 1041.4846 1045.5657 1081.5660 1094.6393 1123.5216 1140.6257 1151.6597 1179.5806 1198.6117 1237.5847 1251.7101 1258.6385 1264.6728 1267.6759 1277.6951 1294.6699 1311.6514 1337.5819 1360.6477 1367.6365 1375.7053 1424.7336 1475.7213 1496.7138 1510.6506 1535.8694 1555.7990 1589.9143 1613.8034 1624.8305 1664.9408 1668.8845 1700.9492 1717.9895 1761.8855 1765.8807 1781.9631 1794.8734 1881.0306 1940.9391 1994.0829 2015.9654 2076.0592 2107.1025 2211.0999 2225.0950 2240.1006 2292.0774 2298.1665 IEF spot 1689: 1151.6236 1252.6359 1264.6176 1277.6329 1307.6662 1341.7101 1344.6395 1368.5754 1375.6627 1383.6225 1394.6589 1419.6925 1497.7909 1509.7184 1535.8266 1555.7803 1566.7852 1570.8423 1578.7812 1585.8527 1613.8076 1621.7580 1650.8802 1707.7462 1712.7774 1715.8487 1728.8015 1794.7926 1804.8966 1818.9066 1825.8959 1838.8990 1881.0462 1895.9270 1936.9580 1952.9601 1962.9766 1980.9366 2082.9915 2099.0082 2139.9269 2155.9884 2196.2051 2211.0999 2225.1098 2239.1133 2283.1903 2314.2017 2362.0761 2528.2225 IEF spot 1707: 842.5100 1187.5823 1277.6596 1306.7072 1383.6581 1424.7845 1475.7350 1481.8440 1560.7928 1589.8204 1612.7670 1647.8648 1708.8533 1743.8693 1794.8108 1826.7289 1940.9432 1993.9578 2020.0073 2034.9747 2083.9911 2211.1000 IEF spot 1715: 842.5100 995.5831 1009.5829 1023.5820 1045.5202 1070.4854 1087.5118 1154.6259 1314.6766 1316.5599 1370.6312 1427.7708 1593.7765 1675.7657 1713.7559 1794.7779 1900.9901 1940.9405 1981.9982 2211.0999 2225.0993 2230.1190 2239.1017 2353.1943 2447.2427 2455.3168 2465.3108 2664.2730 2721.2672 IEF spot 1766: 842.5100 944.4946 989.5409 1017.6835 1032.5402 1045.5083 1060.5038 1071.5384 1077.0911 1084.5774 1107.4665 1129.5720 1179.5248 1193.5853 1201.5667 1205.5819 1234.6034 1263.6062 1277.6315 1281.5339 1314.7075 1320.5393 1325.6773 1349.6438 1363.6477 1381.7550 1398.6787 1407.6259 1434.7108 1436.6620 1475.7174 1491.8240 1493.6869 1505.8475 1519.8568 1556.8407 1602.7688 1636.8141 1678.8399 1707.7623 1743.8024 1780.7768 1794.7887 1838.9008 1872.9419 1931.1324 1993.9690 2083.0147 2097.9525 2211.1000 3451.8065 IEF spot 1800: 842.5100 965.4350 976.4934 992.4239 1045.5079 1065.4919 1074.4756 1120.5483 1164.5137 1179.5419 1254.6347 1260.5475 1277.6344 1292.6279 1302.6309 1308.6085 1383.6186 1415.5896 1419.6711 1434.7317 1467.8188 1475.7193 1487.7064 1493.6788 1506.7180 1522.7029 1524.6992 1553.6528 1639.8495 1657.7832 1665.7380 1676.7759 1681.7378 1707.7739 1709.7370 1716.8454 1759.9521 1783.7564 1791.8506 1794.8215 1838.9510 1940.9262 1993.9821 2211.1000 2671.4041 2687.4419 2721.3115 IEF spot 1902: 1045.5319 1352.5423 1794.7875 1942.9269 2211.0737 2225.0935 2230.1477 2240.0732 2285.1071 2298.1320 2315.1105 IEF spot 1935: 842.5100 976.5614 995.5965 1045.5409 1137.6845 1277.6514 1320.6409 1411.7267 1437.8481 1445.6587 1463.6928 1475.7462 1515.7311 1540.7581 1592.7102 1794.8039 1878.9334 1940.9419 1993.9565 2211.0999 2225.1032 2230.2255 2239.1103 2284.2093 2293.1057 2298.2019 2406.2187 IEF spot 2041: 842.5100 870.5188 995.5983 1009.5573 1011.5865 1045.5044 1073.4231 1253.5788 1277.6266 1287.5708 1357.6107 1391.6701 1427.6972 1475.7224 1487.7416 1567.7156 1567.7156 1615.7750 1624.7871 1715.8582 1745.9047 1794.7998 1826.7704 1940.9130 1993.9553 2011.9662 2082.9776 2211.0999 2298.1935 IEF spot 2079: 842.5100 1045.5126 1074.5097 1128.4836 1144.4893 1179.5318 1201.6130 1215.4643 1226.5814 1232.5216 1267.6287 1272.5458 1277.6236 1291.6468 1314.6885 1320.5261 1330.5987 1350.6193 1475.6943 1535.8375 1553.7165 1624.8774 1649.7828 1657.7624 1664.9078 1687.9333 1707.7469 1791.7278 1794.8045 1813.8890 1826.7495 1838.9154 1851.9083 1940.8912 1970.0934 1991.1016 2096.9820 2150.0510 2198.9812 2211.0999 2225.1041 2230.2625 2239.1553 2246.2206 2314.1744 2338.1612 2367.2374 2383.9340 2501.2666 2617.5910 3121.5635 3126.5388 IEF spot 2354: 842.5100 870.4939 1012.5286 1022.5067 1029.3960 1045.4719 1056.5117 1073.4775 1081.4615 1090.4351 1094.5511 1102.4539 1128.4784 1140.5488 1151.5872 1157.5311 1179.4996 1191.5849 1194.5774 1198.5323 1234.5742 1251.6062 1259.5414 1263.5214 1267.6025 1273.4958 1277.5635 1300.4431 1307.5481 1313.6037 1320.4906 1340.5876 1364.6104 1364.6104 1375.6429 1383.5667 1388.6014 1402.6673 1434.6889 1474.6751 1493.6490 1496.6232 1535.8077 1555.7629 1577.6201 1604.7703 1613.7706 1664.8756 1667.8486 1692.8624 1707.7665 1716.7989 1735.7390 1777.9508 1781.8353 1794.7863 1804.9251 1825.6777 1838.8740 1851.8978 1881.0424 1889.9527 1910.9100 1916.8051 1940.8769 1966.0780 1982.9535 1991.0975 1994.0892 2076.0763 2112.0231 2211.0999 2225.1186 2230.1505 2239.1165 2283.1613 2292.1405 2297.1707 IEF spot 2382: 792.5768 810.5959 814.6006 824.2747 830.5626 842.5100 877.0198 889.9431 905.6620 923.6817 927.6588 943.6340 1002.9798 1018.7167 1036.7046 1040.7238 1056.6945 1063.3297 1079.0752 1116.0163 1131.7560 1149.7860 1153.7654 1169.7402 1229.0710 1244.8292 1262.8523 1266.8593 1282.7904 1342.0994 1357.9284 1375.9517 1379.9293 1413.9492 1471.0408 1475.6861 1489.0704 1493.0714 1584.1187 1602.1516 1697.2401 1715.2577 1737.1939 1828.3534 1993.9914 2211.1000 2225.1434 IEF spot 2408: 842.5100 1045.4988 1239.5138 1287.6241 1462.7030 1590.8423 1601.7867 1794.7912 1838.8872 1903.0143 1940.9054 1987.1174 1993.9468 2083.0461 2211.1000 IEF spot 2411: 842.5100 870.5059 895.4053 995.5448 1009.5518 1023.5688 1045.4862 1179.4954 1201.5783 1234.5657 1263.5824 1277.6078 1307.5765 1314.6757 1325.6195 1383.6090 1404.5953 1427.7396 1434.6573 1458.6092 1460.6883 1462.6815 1475.6602 1478.6293 1512.7909 1524.8316 1564.6905 1576.6922 1580.6980 1611.7268 1638.7754 1649.7423 1660.7930 1700.7628 1707.7236 1716.7952 1725.7846 1743.8049 1754.9104 1762.7919 1766.7505 1794.7743 1826.7847 1837.8701 1855.8577 1900.9779 1940.9038 1993.9091 2011.9112 2016.0210 2027.1124 2082.9987 2152.9815 2196.1547 2201.1245 2211.1000 2284.1674 2298.1775 2315.1778 2402.2727 2521.3566 IEF spot 13652: 973.5179 1036.4774 1060.5342 1065.4002 1090.4578 1157.5122 1179.5299 1193.5473 1263.6227 1277.6329 1307.6100 1320.5257 1383.6186 1421.6125 1434.6909 1458.6522 1475.6960 1493.6697 1513.7133 1532.7183 1638.8153 1657.7778 1707.7530 1716.8296 1784.6837 1796.9843 1837.9545 1851.9192 1941.9522 1993.9786 2054.1177 2150.1636 2184.0966 2211.0999 2225.1274 2383.9529 2400.0149 2510.1564 IEF spot 14098: 1151.6250 1187.5380 1211.6015 1252.6166 1263.6201 1267.6316 1277.6330 1302.6286 1307.6252 1329.6415 1340.5773 1383.6123 1424.6965 1460.7071 1475.6824 1487.7308 1535.8328 1555.7827 1589.7920 1613.8149 1638.8404 1647.7635 1663.3717 1700.9248 1707.7385 1716.8250 1743.8547 1778.9302 1794.7865 1837.9159 1851.9112 1872.9419 1881.0100 1922.9211 1933.9521 1940.9217 1964.9583 1987.0662 1993.9735 2035.0110 2082.9785 2184.1098 2196.1297 2211.0999 2225.1263 2230.1914 2239.1200 2250.0874 2314.1757

TABLE 7 HUMAN ISLET CELL PROTEINS CHARACTERISED BY MS The peptide molecular weight values are read directly out of the mass spectrometer and are thus associated with the accuracies normally obtained with such instrumentation. These mass values are from human islet cell proteins recovered from the NEPHGE gels and do not match any protein recorded in publicly available databases (whether nucleotide or protein sequence). NEPHGE spot 26: 842.5131 870.5369 1045.5217 1179.5371 1265.5405 1277.6271 1308.5457 1314.6746 1320.5417 1324.5497 1357.6471 1427.7444 1475.6748 1493.6661 1664.8418 1703.9330 1716.7841 1719.8587 1743.8199 1760.7887 1775.8273 1791.7874 1794.7814 1826.7590 1940.8930 1945.9745 1993.9401 2211.0453 2225.0987 2230.1019 2239.1267 2284.1508 2299.1526 2314.0976 2383.8783 NEPHGE spot 35: 1169.6652 1277.6525 1320.5251 1366.6936 1403.7316 1427.7965 1467.6493 1475.7490 1486.6881 1550.7053 1638.8392 1651.8001 1707.8210 1794.8089 1838.8934 1940.9146 1950.9534 1994.0184 2011.9465 2082.9935 2152.1050 2157.1118 2211.0999 2292.1087 2313.1264 2368.9793 2418.3266 2439.1478 2467.3066 2468.3029 2563.0697 2581.4875 2597.4212 2921.4292 3034.4707 3220.5008 3494.9136 3510.9276 3636.8963 NEPHGE spot 60: 842.5164 1277.6644 1302.6646 1360.5887 1383.6568 1385.7016 1393.6752 1401.7137 1438.8428 1475.7287 1553.7800 1624.9132 1638.9032 1662.9161 1667.8327 1707.8457 1716.8488 1747.9246 1751.9333 1774.9206 1794.8637 1816.8897 1837.9933 1841.0036 1851.9912 1941.0238 1994.0339 2150.1644 2211.1608 NEPHGE spot 76: 1383.6718 1446.7509 1475.7966 1670.8364 1675.8901 1708.8213 1794.8184 1940.8887 1962.9070 1993.9970 2082.9848 2153.0730 2211.0999 2225.1123 2230.1870 2239.1271 2280.1950 2283.2022 2292.0475 2297.1802 2344.2374 2401.2168 2663.2692 2720.2978 2748.2944 2807.3426 2914.5394 2921.4777 3034.4733 3121.4973 3494.8696 NEPHGE spot 85: 1045.5675 1106.4748 1179.5952 1195.5825 1277.6749 1308.6332 1320.5991 1350.6556 1475.7490 1574.8568 1638.8508 1649.8395 1657.8089 1687.9355 1707.7763 1716.8866 1794.8259 1813.8380 1838.9120 1901.0576 1940.9125 1993.9837 2016.0573 2024.0827 2083.9875 2121.0337 2153.0677 2211.1000 2298.1652 NEPHGE spot 128: 842.5173 950.6940 985.5799 1011.6137 1033.7815 1045.5060 1068.6652 1077.7487 1179.5300 1211.6160 1277.6031 1299.8620 1307.6073 1334.7733 1335.7890 1338.7442 1341.9049 1370.7179 1376.8093 1425.0543 1434.6812 1541.0283 1665.9233 1691.1798 1726.0926 1727.1155 1761.0134 2211.0275 NEPHGE spot 130: 1045.5140 1179.5478 1193.5777 1277.6468 1307.6332 1338.7790 1365.5711 1383.6908 1389.6799 1434.7248 1475.7414 1493.7520 1535.8696 1666.0050 1707.8047 1716.9037 1743.9190 1794.8584 1838.0415 1994.0216 2035.0458 2211.1738 NEPHGE spot 171: 908.7717 942.7243 944.7402 950.8137 1033.8325 1045.6096 1060.5907 1157.5914 1165.5494 1179.6000 1201.6932 1232.5837 1235.5335 1277.7091 1291.6390 1299.9770 1308.6576 1327.7262 1333.9192 1335.9331 1342.0107 1370.6904 1393.7220 1444.8475 1461.7450 1475.7837 1493.7477 1533.7963 1567.8593 1581.7525 1586.7939 1599.8235 1605.8154 1616.8242 1629.8415 1657.8337 1682.8824 1691.2433 1707.8322 1714.8844 1716.8714 1741.7966 1744.9603 1752.8348 1791.7680 2099.0363 2211.0999 NEPHGE spots 187 and 188: 1123.5817 1251.7002 1292.6499 1403.7788 1448.7823 1508.7700 1576.8817 1638.9820 1687.9971 1780.0016 1908.0271 2002.1204 2182.3150 2199.3031 2211.2807 2225.2657 2230.3124 2283.2795 2295.3212 2587.5794 2662.7369 2705.3950 2720.5340 2748.5163 NEPHGE spot 195: 973.5609 976.5443 1045.5840 1157.5838 1179.5999 1201.6708 1234.6620 1263.6625 1277.7208 1284.6043 1300.5175 1307.6708 1320.5245 1323.6305 1337.6572 1357.6867 1365.6012 1383.7097 1386.6658 1434.7588 1475.7479 1493.7223 1638.8590 1707.7833 1716.8781 1755.8142 1794.7931 1837.9626 1851.9484 1882.0046 1890.9783 1905.0450 1940.9194 1993.9950 2083.0237 2211.0999 NEPHGE spot 243: 1045.5466 1060.0443 1066.0453 1794.7869 2211.1470 2225.1316 2236.2563 2239.2134 NEPHGE spot 270: 950.7789 973.5897 989.5751 1003.5955 1025.6590 1029.6033 1036.5737 1045.5866 1060.5666 1064.6402 1074.5264 1082.5967 1090.5566 1107.5747 1157.5786 1165.5700 1179.6000 1201.6640 1213.6248 1232.6071 1234.6742 1257.6697 1259.6823 1263.6743 1277.7118 1299.9629 1300.5490 1307.6720 1323.6470 1335.9371 1357.7143 1379.7259 1383.6952 1393.7250 1404.7544 1407.6986 1434.7663 1438.7044 1475.7611 1493.7269 1504.8006 1523.8004 1560.8202 1638.9009 1657.7832 1691.2608 1707.7958 1716.8609 1791.7356 1793.9708 1795.8320 1837.9885 1851.9594 1890.9722 1940.9413 1993.9967 2054.1412 2064.1473 2086.0239 2120.0246 2211.0999 2509.1782 NEPHGE spot 421: 1045.5551 1140.6401 1179.5911 1201.6178 1218.6234 1233.6811 1277.6552 1302.6608 1307.6456 1314.7202 1454.6592 1475.7489 1535.8938 1560.7882 1567.8222 1628.7809 1660.8150 1664.9552 1716.8609 1784.9210 1793.1088 1913.0411 1940.9146 1963.9912 1993.9933 2211.0999 NEPHGE spot 449: 1775.7252 2210.8860 2224.9665 2238.8424 2663.1016 2720.1628 2749.1314 2807.1013 2821.1104 3121.5130 NEPHGE spot 508: 842.5100 1045.5536 1066.5542 1179.5953 1183.6831 1236.5941 1291.6831 1295.6332 1307.6875 1403.6576 1428.8009 1475.7001 1665.6731 1763.8092 1784.9099 1794.8168 1801.9166 1816.9433 1993.9722 2052.1099 2072.1142 2140.0546 2180.2004 2211.0999 2225.1141 2230.1947 2239.1373 2246.1973 2283.1010 2292.0967 2299.1391 2313.1951 2344.1912 2361.2462 2663.3861 2678.1476 2720.3396 2748.1750 2807.3801 2821.3286 3121.6227 3339.9020 3349.7006 NEPHGE spot 509: 842.5661 950.7645 1045.5750 1138.4976 1141.5052 1157.5242 1162.5323 1179.5556 1188.6617 1192.5322 1201.6627 1206.5864 1207.5732 1231.5848 1248.5986 1254.5988 1264.6176 1277.6566 1299.9226 1308.6124 1309.6189 1314.7051 1335.8741 1341.9762 1351.6377 1369.6827 1408.7250 1431.6785 1440.6923 1475.7310 1548.7768 1562.7803 1582.8090 1621.9304 1691.2267 1716.8358 1725.1946 1727.1917 1749.9958 1794.8245 1990.0044 2006.0106 2034.9689 2211.1000 2398.2911 2672.2803 3530.7912 NEPHGE spot 532: 842.5100 856.5163 987.4343 998.4821 1012.4873 1045.5354 1066.5093 1078.4847 1080.5039 1092.4997 1100.5099 1665.7157 1679.7342 1691.7426 1697.7075 1707.7815 1794.8100 1826.7846 1846.8073 1940.9527 2011.9847 2078.1252 2082.9893 2211.0999 2225.0967 2239.1366 2283.1418 2297.1983 2313.2183 2344.2588 2663.3824 2691.3461 2705.3164 2720.3205 2734.3788 2744.3915 2748.3478 2807.3461 2821.4211 2914.6472 2921.3275 3338.9020 3348.7397 NEPHGE spot 558: 831.5173 908.6378 944.5866 950.6773 973.4943 1003.4784 1027.4818 1031.5106 1033.4303 1037.4838 1045.4983 1090.4512 1118.4363 1157.5146 1165.4922 1179.5187 1193.5475 1201.5905 1232.5216 1234.6120 1254.6082 1277.6373 1300.4872 1302.6032 1308.5774 1320.5401 1340.5812 1357.6289 1365.5703 1383.6340 1393.6433 1418.6599 1434.6912 1443.7196 1454.6651 1475.7090 1493.6847 1560.7731 1608.7220 1657.7727 1707.7505 1716.8222 1791.7034 1794.7971 1838.9112 2138.1215 2211.1154 2533.4148 NEPHGE spot 560: 1678.8305 1700.8144 1732.7947 1760.8185 2211.1243 NEPHGE spot 609: 842.5823 1045.6003 1074.5479 1111.6290 1179.6000 1232.5793 1307.6391 1320.5689 1350.6723 1385.7186 1475.7164 1479.7159 1493.7217 1503.7389 1548.8342 1573.7621 1638.8672 1650.9216 1657.8110 1707.7865 1743.8224 1791.7538 1794.7899 1813.9147 1826.8037 1838.9318 1851.9457 1867.9339 1994.0167 2091.9105 2150.1387 2198.9669 2211.1000 2383.9361 2501.2078 2717.0710 NEPHGE spot 719: 1245.4899 1256.5258 1273.5880 1600.6902 1738.8842 1743.7380 1794.7060 2034.7818 2210.8631 2282.9633 2541.9943 2637.0529 2662.1501 2719.9902 2780.1056 NEPHGE spot 729: 842.6014 973.5732 995.6290 997.6764 1009.6652 1011.6854 1037.5903 1045.5939 1060.6033 1140.5753 1157.5940 1165.6028 1179.6000 1234.6787 1263.6654 1277.7022 1300.5221 1307.6667 1314.7520 1320.5957 1323.6536 1357.6990 1379.7386 1383.6807 1427.8123 1434.7499 1475.7565 1487.7571 1493.7502 1638.8667 1699.8308 1707.7936 1716.8692 1784.7536 1794.8321 1837.9871 1851.9540 1993.9841 2211.0999 NEPHGE spot 829: 728.6920 754.5651 806.5817 840.6673 901.6037 908.7999 922.8081 929.5580 942.7504 944.7633 950.8350 984.6868 1045.6162 1060.5888 1065.5604 1069.5700 1101.6517 1107.5696 1115.5327 1168.6149 1175.6590 1179.6000 1191.6387 1201.6858 1204.6558 1234.6775 1264.5820 1277.7048 1289.6860 1297.7003 1307.6572 1314.7802 1335.9463 1344.7647 1357.7094 1383.6713 1475.7766 1599.8000 1641.7130 1716.8676 1743.8138 1759.8716 1761.8461 1763.9059 1778.9217 1785.9446 1794.8405 2211.0999 3553.7439 NEPHGE spot 836: 550.6338 559.4159 594.3097 620.3219 628.2701 634.3534 677.3904 705.4059 719.4081 763.4069 777.3504 816.4305 842.5100 856.5303 870.5226 901.4988 944.5088 953.5746 967.5807 979.4747 985.5182 1020.4115 1032.5642 1045.5452 1060.5229 1088.5526 1107.5244 1126.4778 1137.4690 1151.4924 1168.5205 1179.5371 1217.5778 1265.5486 1277.6444 1308.6107 1314.6961 1320.5574 1323.6041 1334.6886 1349.7357 1369.6180 1377.7381 1404.5148 1411.6379 1475.7199 1493.7304 1504.8048 1664.9080 1703.9596 1719.9754 1733.9201 1736.0145 1743.7900 1759.7683 1775.8072 1791.8303 1794.8143 1806.8917 1826.8090 1831.7637 1837.9212 1853.9619 1858.9394 1906.9335 1945.9915 1993.9626 2113.0476 2117.9938 2123.1281 2128.0641 2211.1000 2221.1253 2225.1040 2240.1259 2283.1314 2292.0664 2299.1709 2314.1811 2383.9977 NEPHGE spot 837: 842.5100 944.5317 1020.4700 1055.5366 1242.5797 1281.5708 1362.6911 1625.7170 1763.7852 1794.7933 2152.9836 2211.1000 3173.9268 NEPHGE spot 3879: 806.4919 1029.6369 1036.5549 1045.5927 1065.5397 1087.5387 1090.5970 1115.5180 1157.6195 1165.5772 1175.6512 1179.6000 1198.6571 1201.6794 1205.6320 1232.6643 1243.6040 1277.7034 1297.6864 1300.5622 1307.6610 1314.7621 1320.5558 1323.6573 1330.7207 1337.6185 1340.7602 1344.7320 1383.6828 1426.7184 1434.7529 1438.8223 1441.7131 1469.7683 1475.7527 1493.7449 1535.8745 1634.8796 1638.8827 1656.7456 1675.8037 1707.7860 1716.8731 1728.8562 1743.8410 1759.8798 1785.9333 1794.8294 1804.8441 1838.9205 1990.1067 1994.0040 2211.1000 NEPHGE spot 6600: 1112.7125 1119.8876 1154.7604 1179.5853 1258.5560 1299.9739 1334.8759 1342.0311 1369.7625 1376.9353 1404.6771 1411.8281 1691.3292 1726.2245 1761.1320 1794.8996 2101.1599 2117.5243 2211.2240

TABLE 8 PROTEINS DETECTED IN HUMAN ISLET CELL MEDIA AND CHARACTERISED BY MS The peptide molecular weight values are read directly out of the mass spectrometer and are thus associated with the accuracy normally obtained with such instrumentation. These mass values are from proteins detected in the media following incubation of human islets in growth media (as described in the methods). The proteins are recovered from the IEF gels. Their peptide mass values do not match to the peptides which would be generated if any protein, recorded in publicly available databases (whether nucleotide or protein sequence), was treated in a similar manner. IEF spot 122SPI: 842.5100 927.4963 973.5343 1066.4649 1109.4248 1165.5350 1179.5583 1213.6268 1234.6228 1263.6294 1277.6595 1300.4708 1307.6346 1315.6246 1320.5471 1357.6533 1365.5835 1379.6918 1383.6430 1390.6393 1400.6369 1405.6680 1421.6233 1427.7104 1434.7311 1475.7333 1493.6983 1497.7529 1513.7643 1639.9035 1657.7697 1699.8245 1707.7840 1716.8404 1745.8399 1797.0431 1819.0044 1834.9854 1837.9651 1993.9989 2096.1253 2114.1074 2129.9106 2211.0999 2224.2375 2239.1053 2249.0984 2367.2937 2383.9584 2509.2311 2705.2275 2872.5248 3314.3928 IEF spot 123SPI: 842.5100 995.6139 1045.5020 1179.5325 1257.6289 1277.6396 1307.6108 1382.6104 1418.6783 1434.7291 1475.7101 1487.7178 1523.7513 1540.7742 1604.7757 1608.7595 1638.8370 1707.7398 1716.8473 1794.8201 1812.0723 1837.9388 1851.9256 1867.9182 1901.0100 1940.9715 1993.9586 2113.0096 2150.0818 2184.0996 2211.0999 2225.1103 2231.1825 2345.2339 2384.9304 IEF spot 126SPI: 842.5100 1023.6778 1095.6006 1137.7430 1164.6686 1198.6274 1204.6551 1217.6581 1277.6826 1291.6875 1308.6616 1313.6816 1366.7194 1371.7416 1383.6704 1428.7966 1439.7924 1444.7147 1475.7456 1487.7390 1570.8290 1619.8479 1629.8740 1638.8514 1662.8514 1674.8398 1679.8136 1684.8887 1692.8810 1707.7897 1794.8050 1824.9642 1837.9740 1852.9313 1867.9291 1869.9953 1896.0050 1902.0257 1940.9322 1953.0459 1962.9743 1993.9616 2151.0562 2158.1156 2169.0372 2184.0868 2211.1000 2283.2157 2297.1638 2345.2835 2748.3837 IEF spot 130SPI: 842.5100 1036.5147 1075.5384 1082.5560 1107.5100 1165.5090 1179.5516 1198.6566 1277.6451 1303.6580 1307.6217 1329.6573 1393.6412 1455.6351 1475.7144 1487.7050 1515.7538 1638.8218 1657.7565 1704.8650 1707.7353 1758.8640 1790.8768 1794.7723 1799.8790 1811.8747 1837.9550 1839.9159 1872.9456 1914.0100 1941.9548 1993.9509 2047.0529 2054.0744 2150.0295 2184.0963 2211.0999 2250.0364 2383.9437 IEF spot 135SPI: 842.5100 1271.6481 1343.6065 1413.7382 1440.6092 1452.6148 1515.7276 1571.7985 1692.8303 1709.7921 1794.8135 2211.1000 IEF spot 140SPI: 842.5100 995.6118 1192.5588 1198.6323 1277.6308 1311.5802 1333.6658 1344.5990 1350.6185 1419.6963 1475.7224 1487.6666 1515.7236 1575.7435 1638.8524 1687.8674 1707.7437 1773.8513 1780.8198 1790.8845 1794.8104 1838.9057 1853.9362 1858.9484 1901.1178 1954.0130 1993.9445 2016.0588 2075.9670 2155.1313 2211.0999 2279.2190 2284.2035 2384.9802 2401.1453 IEF spot 160SPI: 842.5100 944.4201 1045.4319 1093.3863 1121.4125 1179.4628 1195.4396 1277.5655 1297.5044 1302.5312 1307.5412 1320.4609 1350.5468 1383.5719 1475.6543 1556.6594 1638.8052 1650.7700 1657.7602 1684.7837 1707.7410 1794.7624 1812.8635 1838.9208 1851.8857 1872.9481 1931.9711 1993.9814 2211.0999 2225.1307 2383.9509 2389.2171 IEF spot 218SPI: 842.5100 950.6771 995.4745 1021.4311 1043.4929 1045.4833 1060.5128 1109.4391 1126.4305 1142.4871 1157.5613 1179.5079 1199.4923 1210.5203 1220.5043 1234.6055 1263.6086 1277.6299 1286.5597 1300.4350 1302.6175 1307.5960 1314.7119 1320.5066 1329.5849 1353.6598 1357.6076 1365.5460 1383.5880 1393.6262 1427.7609 1434.7341 1475.6921 1493.6654 1545.7737 1553.7960 1700.7823 1707.7831 1714.8079 1716.8088 1726.8174 1794.7979 1825.9672 1838.9224 1922.9490 1933.9836 1940.9304 1987.0506 1993.9688 2005.0097 2083.0669 2211.0999 2225.1217 2230.1991 2239.1338 2283.2517 2297.1804 2344.3708 2367.2739 2384.1822 2401.2136 2662.6332 3077.6726 3095.4811 3339.9445 3349.7509 IEF spot 248SPI: 842.5100 995.5661 1192.5533 1198.6455 1344.5956 1419.6789 1424.7739 1515.7473 1704.8844 1794.8113 1869.9654 1902.0689 1940.9132 1952.9542 1959.9920 2016.1083 2153.1172 2155.0712 2211.1000 2279.1779 2284.1800 2344.2439 2402.2584 IEF spot 277SPI: 842.5100 944.5108 989.5777 1032.5590 1137.6793 1180.5632 1277.6210 1325.6529 1335.6512 1349.6238 1363.6523 1366.7033 1398.7028 1467.7336 1475.7235 1538.8441 1602.7634 1636.8005 1638.8129 1652.7675 1670.9066 1703.8669 1719.9301 1743.8032 1758.8949 1794.7991 1839.8938 1931.1572 1940.9432 1993.9414 2030.0920 2153.0395 2176.9849 2211.0999 2284.1637 2298.1739 2345.2744 IEF spot 304SPI: 842.7320 2211.6199 2225.6395 2284.7376 2346.7681 IEF spot 314SPI: 1747.1466 1929.2249 2040.3318 2079.2340 2292.4766 2309.5383 2338.5453 2367.6507 IEF spot 338SPI: 842.5100 1277.6344 1298.6552 1302.6274 1307.5972 1329.6478 1381.7755 1398.6603 1426.7642 1475.7128 1487.6784 1523.7645 1602.7505 1638.8279 1707.7565 1742.8419 1758.8799 1784.7091 1794.7915 1812.0358 1838.9245 1851.9135 1867.8992 1941.9594 1966.0151 1969.0193 1993.9537 2150.0574 2176.0349 2184.0894 2211.0999 2225.1184 2286.1485 2299.1497 2331.1452 2361.1860 2383.9924 2501.1805 2509.2701 2580.3195 2643.3333 2702.2667 2707.2342 2718.2130 2723.3186 2808.3617 2832.3462 

1. A method for diagnosing diabetes in a mammal, the method comprising determining in a pancreas sample the level of expression of at least one marker protein selected from the group consisting of: a) P05451 (known as islet cell regeneration factor; lithostathine 1 alpha, pancreatic stone protein; and pancreatic thread protein); b) P 19971 (known as thymidine phosphorylase; platelet derived endothelial cell growth factor; and gliostatin); c) P30740 (known as leukocyte elastase inhibitor); and d) P80188 (known as alpha 2 microglobulin related subunit of MMP and lipocalin-2), wherein increased expression of a), b), c) and/or d) indicates that the mammal has diabetes.
 2. The method according to claim 1, wherein the mammal is a human.
 3. A method for determining the predisposition for diabetes in a mammal, the method comprising determining in a pancreas sample the level of expression of at least one marker protein selected from the group consisting of: a) P05451 (known as islet cell regeneration factor; lithostathine 1 alpha, pancreatic stone protein; and pancreatic thread protein); b) P19971 (known as thymidine phosphorylase; platelet derived endothelial cell growth factor; and gliostatin), c) P30740 (known as leukocyte elastase inhibitor); and d) PS0188 (known as alpha 2 microglobulin related subunit of MMP and lipocalin-2), wherein increased expression of a), b), c) and/or d) indicates that the mammal has a predisposition for diabetes.
 4. A method for determining the predisposition in a human for diabetes, the method comprising: i) determining in a pancreas sample originating form the human the increased expression of at least one marker protein selected from the group consisting of: a) P05451 (known as islet cell regeneration factor; lithostathine 1 alpha, pancreatic stone protein; and pancreatic thread protein); b) P19971 (known as thymidine phosphorylase; platelet derived endothelial cell growth factor; and gliostatin); c) P30740 (known as leukocyte elastase inhibitor); and d) P80188 (known as alpha 2 microglobulin related subunit of MMP and lipocalin-2), wherein increased expression of a), b), c) and/or d) indicates that the mammal has a predisposition for diabetes; ii) determining in a pancreas sample originating form the human the decreased expression of at least one marker protein selected from the group consisting of: a) P05451 (known as islet cell regeneration factor; lithostathine 1 alpha, pancreatic stone protein; and pancreatic thread protein); b) P 19971 (known as thymidine phosphorylase; platelet derived endothelial cell growth factor; and gliostatin); c) P30740 (known as leukocyte elastase inhibitor); and d) P80188 (known as alpha 2 microglobulin related subunit of MMP and lipocalin-2), wherein decreased expression of a), b), c) and/or d) indicates that the mammal has a reduced predisposition for diabetes. 